Methods and products related to metabolic interactions in disease

ABSTRACT

The invention involves methods of regulating cell growth and division to control disease processes by manipulating mitochondrial metabolism and the expression of cell surface immune proteins. The invention also involves related compositions and screening assays.

RELATED APPLICATIONS

This application is a continuation of application of Ser. No.09/711,022, filed Nov. 9, 2000, now pending, which is a continuation ofSer. No. 09/277,575, filed Mar. 27, 1999, entitled METHODS AND PRODUCTSRELATED TO METABOLIC INTERACTIONS IN DISEASE, and now pending, whichclaims priority to U.S. Provisional application Ser. No. 60/082,250,filed Apr. 17, 1998; 60/101,580, filed Sep. 24, 1998; and 60/094,519,filed Jul. 29, 1998, now abandoned, each of which the entire contentsare incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant/Contract No.AI 33470 awarded by the National Institute of Health. The Government mayretain certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to methods of regulating cell growth and divisionto control disease processes by manipulating mitochondrial metabolismand the expression of cell surface immune proteins. The invention alsorelates to compositions and screening assays.

BACKGROUND OF THE INVENTION

Normal tissue develops and is maintained by normal processes of celldivision and cell death. In many diseases, such as cancer, diabetesmellitus Type I, and autoimmune disease, the normal balance between celldivision and cell death is disrupted causing either a rapid growth ofunwanted and potentially dangerous cells or a loss of cells which areessential to maintaining the functions of tissue.

Cell division occurs by a process known as mitosis. During mitosisdividing cells use glucose cytolytically at an increased rate as theprimary source for energy (ATP) production in a process referred to asglycolysis (Brand, K. A., and U. Hermfisse. 1997. Aerobic glycolysis byproliferating cells: a protective strategy against reactive oxygenspecies. Faseb J 11, no. 5:388-95). Glycolysis occurs in the cytosol andis required for mitochondrial energy production. An increased rate ofglycolysis occurs when cells divide, providing more of the ATP fromcytosolic glycolysis. Mitochondrial synthesis of ATP proceeds throughcoupling of electron transport-dependent oxido-reductive reactions toATP synthetase (oxidative phosphorylation) (Harper, M. E. 1997. Obesityresearch continues to spring leaks. Clinical Investigations in Medicine20, no. 4:239-244). During this process, a proton gradient is generatedby the pumping of protons out of the mitochondria (Himms-Hagen, J. 1992.Brown Adipose Tissue. Obesity, eds. P. Bjorntorp and B. N. Brodoff. 1vols. J. B. Lippincott, Philadelphia. 1 pp), increasing mitochondrialmembrane potential. Uncoupling proteins (UCPs) reversibly uncoupleoxidative phosphorylation from electron transport and thereby candecrease mitochondrial membrane potential (Harper, M. E. 1997. Obesityresearch continues to spring leaks. Clinical Investigations in Medicine20, no. 4:239-244). Elevating glucose concentrations can increasemitochondrial membrane potential (Harper, M. E. 1997. Obesity researchcontinues to spring leaks. Clinical Investigations in Medicine 20, no.4:239-244).

Cell death is a physiologic process that ensures homeostasis ismaintained between cell production and cell turnover in self-renewingtissues and is essential to the proper functioning of the immune system.Physiological cell death occurs through the processes of apoptosis andnecrosis. The boundaries between these processes, once thought to bedistinct, have blurred with the explosion of information on the role ofcell death in development, tissue modeling, regenerative processes, andin the immune system. However, it is widely accepted that necrotic celldeath (sometimes called oncosis) typically results in the osmoticrupture of a cell, followed by an inflammatory response, while apoptoticdeath involves cell shrinkage, fragmentation of the cell, andphagocytosis of the fragments often without inflammation. Most cells diein a form of suicide characteristically apoptotic and tightly regulatedby complex signals (Zakeri, Z., W. Bursch, M. Tenniswood, and R. A.Lockshin. 1995. Cell Death: Programmed, apoptosis, necrosis, or other.Cell Death and Differentiation 2:87-96). Apoptotic cell death isparticularly important in the reticulo-endothelial system where thebalance between mitosis and cell death may determine the effectivenessand the nature of an immune response (Zakeri, Z., W. Bursch, M.Tenniswood, and R. A. Lockshin. 1995. Cell Death: Programmed, apoptosis,necrosis, or other. Cell Death and Differentiation 2:87-96). Failureresults in autoimmune disease or in a lack of immune surveillance.

Inappropriate cell division or cell death results in seriouslife-threatening diseases. Diseases associated with increased celldivision include cancer and atherosclerosis. Disease resulting fromincreased cell death include AIDS, neurodegenerative diseases (e.g.,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa), aplastic anemia, atherosclerosis (e.g.,myocardial infarction, stroke, reperfusion injury), and toxin inducedliver disease. Many methods for treating these disorders have beenproposed Although these diseases share the common physiological trait ofeither excess cell division or premature cell death, strategies foridentifying potential therapeutic treatments have been individualizedrather than searching for a common mechanism. It would be desirable toidentify a common mechanism by which cell division could be interruptedor cell death could be promoted to treat all of these diseases.

PC12 cells, a cell line derived from rat pheochromocytoma (Greene andTischler, 1976) have been extensively used as a model for the study ofnerve growth factor (NGF)-induced neuronal differentiation anddependency (Mills et al., 1997), and of neuronal cell apoptosisresulting from serum and/or trophic factor withdrawal (Mesner et al.,1995, Fulle et al., 1997), oxidative stress (Vinard et al., 1996) and,the addition of calcium ionophores (Fulle et al, 1997). NGF promotesdifferentiation, neurite outgrowth and the acquisition of a maturesympathetic neuronal morphology on PC12 cells. Withdrawal of NGF,however, results in apoptosis of the PC12 cells which is characterizedby prototypic changes, i.e., chromatin degradation, nuclearfragmentation, acidification, alterations of surface lipids, cellfragmentation, blebbing and nucleosome formation (Gottlieb et al, 1997).

PC12 transfected variants such as TrkA have been developed to elucidatethe role of NGF and signal transduction in neuronal function. Nervegrowth factor (NGF) binds to two synergistic receptors, tyrosine kinaseA (TrkA) and p75NGRF (Canossa et al., 1996). The PC12 TrkA cell lineoverexpresses TrkA, a 140 kDa protein with intrinsic tyrosine kinaseactivity (Kaplan et al., 1991) and responds more vigorously than nativePC12 cells to NGF stimulation. It is believed that the NGF-TrkA complexacts as a messenger that delivers the growth signal from axon terminalsto sympathetic neuronal cell bodies (Riccio et al., 1997).

Epidermal growth factor (EGF) has different effects on PC12 cells thanNGF. When tenative PC12 cells are treated with EGF they are induced toundergo proliferation rather than differentiation. In contrast, EGFstimulation of the v-Crk and TrkA cell lines induce neuronaldifferentiation (Teng et al., 1995).

Fas, a member of the tumor necrosis receptor family that includes thenerve growth factor receptor, mediates apoptotic cell death in severalinstances, including TCR (T cell receptor)/CD3-induced T cell activation(Nagata et. al., Science). When the Fas molecule interacts with Fasligand or an appropriate anti-Fas antibody, cellular death can ensue(Gottlieb et al., 1997). Fas was originally described on the membranesurface of hematopoietic lineage cells (Itoh et al., 1991), but itspresence has been documented on endothelial cells (Richardson et al.,1994), hepatocytes (Tanaka et al., 1998) and oligodendrocytes inmultiple sclerosis lesions (Bonetti and Raine, 1997).

The B7 molecules, B7.1 (CD80) and B7.2 (CD86) are known for theirability to co-stimulate T cell proliferation (Linsley et al., 1991), theproduction of interleukin-2 (Freeman et al., 1992) and the expression ofinterleukin-2 receptors (Razi-Wolf et al., 1996). Expression of theseco-stimulatory molecules on immune cells also may play an important rolein the pathogenesis and response to several bacterial, parasitic andviral infections as well as autoimmune disease (Reiser and Stadecker,1996) such as systemic lupus erythematosus (Folzenlogen et al., 1997),experimental allergic encephalomyelitis (Perrin et al., 1996) and in therejection phase of alloimmune responses (Akalin et al., 1997).

B7.1 and B7.2 are members of the immunoglobulin gene superfamily andinclude a V-like and a C2-like extracellular domain. Although originallydescribed on B cells, B7.1 and B7.2 have also been described onmonocytes, dendritic cells and activated T cells (June et al., 1994).B7.1 (CD80) and particularly B7.2 (CD86) are upregulated on the Blymphocyte surface of patients with systemic lupus erythematosus (SLE)(Folzenlogen at al., 1997).

SUMMARY OF THE INVENTION

The invention involves the finding that mitochondrial metabolism playsan essential role in regulating cellular division and cell deathoccurring in various diseases. It was found according to the inventionthat the status of the cellular proton motor force which can be assessedby the coupling relationship between electron transport and oxidativephosphorylation plays an important role in the signal which determineswhether a cell will undergo cellular division, cellular differentiationor cellular death. This finding has important implications for treatingdiseases associated with excessive cellular division, aberrantdifferentiation, and premature cellular death, e.g., for the treatmentof cancers, autoimmune disease, neurodegenerative diseases, etc.

It was also found according to the invention that the expression ofimmune recognition molecules on the surface of cells is important inregulating the processes of cell division, differentiation and apoptosisoccurring in various diseases. It was discovered, for instance,according to the invention that the expression of immune recognitionmolecules on the surface of a cell correlates with the ability of thecell to undergo differentiation. For instance, upon removal of NGF froma nerve cell, the surface expression of B7 molecules is down regulatedand the nerve cell undergoes apoptosis. The induction kinetics andexpression of Fas, B7.1 and B7.2 molecules on the membrane surface ofdifferentiated PC12 cells and its mutants, and TrkA cells have beenexamined and are described in the Examples below.

The invention includes the discovery that neural differentiation andapoptosis are regulated through interaction of the immune recognitionmolecules on the nerve cell surface with an NGF producing cell thatexpresses the counterpart surface immune recognition molecule, likelyCD28 or CTLA4. The interaction between the nerve cell and the NGFproducing cell causes the NGF producing cell to release NGF into thelocal environment. This NGF then stimulates the nerve cell to undergonerve cell differentiation and innervation.

Several cell surface proteins have previously been identified as celldeath proteins. These proteins are believed to be involved in initiatinga signal which instructs the cell to die. Cell death proteins includefor example Fas/CD95 (Trauth et al., Science 245:301, 1989), tumornecrosis factor receptors, immune cell receptors such as CD40, OX40,CD27 and 4-1BB (Smith et al., Cell 76:959, 1994), and RIP (U.S. Pat. No.5,674,734). These proteins are believed to be important mediators ofcell death. These mediators, however, do not always instruct a cell todie. In some cases these mediators actually instruct a cell to undergocell division. Prior to the instant invention the mechanism causing thedual functionality of these cell death proteins was not understood. Itwas discovered according to the invention, that the intracellularenvironment and particularly the status of the proton motor force andsource of fuel for mitochondrial metabolism determines whetherstimulation of the cell death protein will lead to a signal for death orcell division.

It was also discovered according to the invention that the regulation ofcell surface expression of major histocompatibility complex (MHC) classII and co-stimulatory molecules B7-1 and B7-2 can be manipulated byregulating the intracellular dissipation of proton motor force which canbe assessed in terms of mitochondrial membrane potential. Underconditions of low mitochondrial membrane potential (electron transportand oxidative phosphorylation are uncoupled), cells use non-glucosecarbon sources for mitochondrial oxygen consumption (e.g., fatty acidsor amino acids) and the surface expression of MHC class II andco-stimulatory molecules B7-1 and B7-2 is increased. Under conditions ofhigh mitochondrial membrane potential (electron transport and oxidativephosphorylation are relatively more coupled and glucose is being used asa mitochondrial carbon source) the surface expression of MHC class IIand co-stimulatory molecules B7-1 and B7-2 is decreased.

In one aspect the invention is a method for decreasing mitochondrialmembrane potential in a mammalian cell. The method involves the step ofadministering an MHC class II HLA-DR ligand to the mammalian cell toselectively engage MHC class II HLA-DR on the surface of the cell in anamount effective to decrease mitochondrial membrane potential in themammalian cell, wherein the mammalian cell is not an antigen presentingcell. In one embodiment MHC class II HLA-DR is expressed on the surfaceof the mammalian cell. In another embodiment the method involves thestep of contacting the mammalian cell with an amount of an MHC class IIHLA-DR inducing agent effective to induce the expression of MHC class IIHLA-DR on the surface of the mammalian cell.

The mammalian cell may be any type of cell other than an antigenpresenting cell. In one embodiment the mammalian cell is a tumor cell.Preferable the MHC class II HLA-DR ligand is administered to the tumorcell in vivo in an amount effective for causing cell lysis of the tumorcell. When the mammalian cell is a tumor cell, however, in someembodiments the MHC class II HLA-DR inducing agent does not includeadriamycin and gamma interferon. In other embodiments when the mammaliancell is a tumor cell the MHC class II HLA-DR inducing agent does notinclude adriamycin and gamma interferon.

According to another aspect of the invention a method for decreasingmitochondrial membrane potential in a mammalian cell is provided. Themethod involves the step of contacting the mammalian cell with an amountof an MHC class II HLA-DR inducing agent effective to induce theexpression of MHC class II HLA-DR on the surface of the mammalian cell,wherein the mammalian cell is not an antigen presenting cell.

The invention in another aspect is a method for increasing mitochondrialmembrane potential in a mammalian cell. The method involves the step ofadministering an MHC class II HLA-DP/DQ ligand to the mammalian cell toselectively engage MHC class II HLA-DP/DQ on the surface of the cell inan amount effective to increase mitochondrial membrane potential in themammalian cell. In this aspect of the invention the mammalian cell isnot an antigen presenting cell.

In one embodiment MHC class II HLA-DP/DQ is expressed on the surface ofthe mammalian cell. In another embodiment the invention includes thestep of contacting the mammalian cell with an amount of an MHC class IIHLA-DP/DQ inducing agent effective to induce the expression of MHC classII HLA-DP/DQ on the surface of the mammalian cell.

According to another embodiment the mammalian cell is a pancreatic βcell of a type I diabetic and wherein the MHC class II HLA-DP/DQ ligandis administered to the pancreatic β cell in vivo.

The methods of the invention are useful for inducing cell division, celllysis, cell differentiation and cell apoptosis, depending on themetabolic condition of the cell. In one aspect the invention is a methodfor inducing lysis of a mammalian cell. The method includes the steps ofcontacting the mammalian cell with an amount of an MHC class II HLA-DRinducing agent effective to induce the expression of MHC class II HLA-DRon the surface of the mammalian cell, and contacting the MHC class 11HLA-DR on the surface of the mammalian cell with an amount of an MHCclass II HLA-DR ligand effective for causing lysis of the mammaliancell.

In one embodiment the MHC class II HLA-DR ligand is an endogenous MHCclass II HLA-DR ligand and the step of contacting the mammalian cellwith the MHC class 11H LA-DR ligand is a passive step. In anotherembodiment the step of contacting the mammalian cell with the MHC classII HLA-DR ligand is an active step.

The mammalian cell may be any type of cell other than an antigenpresenting cell. The mammalian cell is a tumor cell in anotherembodiment. Preferably the MHC class II HLA-DR ligand is administered tothe tumor cell in vivo in an amount effective for causing cell lysis ofthe tumor cell. When the mammalian cell is a tumor cell, however, theMHC class II HLA-DR inducing agent does not include adriamycin and gammainterferon.

In one aspect the invention is a method for inducing cell lysis in atumor cell. The method involves the steps of contacting a tumor cellwith an amount of an MHC class II HLA-DR inducing agent effective toinduce the expression of MHC class II HLA-DR on the surface of the tumorcell, and contacting the MHC class II HLA-DR on the surface of the tumorcell with an amount of an MHC class II HLA-DR ligand effective forcausing cell lysis of the tumor cell.

The MHC class II HLA-DR inducing agent is any agent which inducesexpression of MHC class II HLA-DR on a cell surface. Preferably theinducing agent is selected from the group consisting of adriamycin,gamma interferon, bacterial byproducts such as lipopolysaccharides,mycobacterial antigens such as BCG, a UCP expression vector, a TCRαβengagement molecule and a fatty acid. Once the MHC class II HLA-DR isexpressed on the surface of the cell an MHC class II HLA-DR ligand caninteract with the MHC class II HLA-DR and initiate cell lysis.Preferably the MHC class II HLA-DR ligand is selected from the groupconsisting of an anti-MHC class II HLA-DR antibody, CD4 molecules, αβcell receptor molecules, γδ T cell receptor molecules and a MHC class IIHLA-DR binding peptide.

In one embodiment the MHC class II HLA-DR inducing agent and the MHCclass II HLA-DR ligand are administered simultaneously. In anotherembodiment the MHC class II HLA-DR inducing agent and the MHC class IIHLA-DR ligand are administered orally. In yet another embodiment the MHCclass II HLA-DR inducing agent and the MHC class II HLA-DR ligand areadministered locally.

In another aspect the invention is a method for inducing cell lysis in atumor cell by contacting a tumor cell with an amount of an MHC class IIHLA-DR inducing agent effective to induce the expression of MHC class IIHLA-DR on the surface of the tumor cell in the presence of an MHC classII HLA-DR ligand. Preferably the MHC class II HLA-DR ligand is an MHCclass II HLA-DR expressing cell. In one embodiment the inducing agent isselected from the group consisting of adriamycin, gamma interferon,bacterial byproducts such as lipopolysaccharides, mycobacterial antigenssuch as BCG, a UCP expression vector, a TCRαβ engagement molecule and afatty acid.

In another embodiment the MHC class II HLA-DR inducing agent and the MHCclass II HLA-DR ligand are administered orally. In yet anotherembodiment the MHC class II HLA-DR inducing agent and the MHC class IIHLA-DR ligand are administered locally.

According to another aspect of the invention a method for inducingapoptosis in a tumor cell is provided. The method involves the steps ofcontacting a tumor cell with an amount of a metabolic modifying agent,which when exposed to a cell causes coupling of electron transport andoxidative phosphorylation, effective to increase the mitochondrialmembrane potential in the tumor cell, and contacting the tumor cell withan amount of an apoptotic chemotherapeutic agent effective for inducingapoptosis in the tumor cell.

The metabolic modifying agent is added to the tumor cell to inducecoupling of electron transport and oxidative phosphorylation. Preferablythe metabolic modifying agent is selected from the group consisting ofglucose, phorbol myristate acetate in combination with ionomycin, MHCclass II HLA-DP/DQ ligand, GDP, CD40 binding peptide, UCP antisense,dominant negative UCP, sodium acetate, and staurosporine. Once electrontransport is coupled to oxidative phosphorylation, Fas expression isinduced on the cell surface and a apoptotic chemotherapeutic agent canbe added to induce apoptosis of the tumor cell. In one embodiment theapoptotic chemotherapeutic agent is selected from the group consistingof adriamycin, cytarabine, doxorubicin, and methotrexate.

In one embodiment the metabolic modifying agent and the apoptoticchemotherapeutic agent are administered simultaneously. In anotherembodiment the metabolic modifying agent and the apoptoticchemotherapeutic agent are administered orally. In yet anotherembodiment the metabolic modifying agent and the apoptoticchemotherapeutic agent are administered locally.

In one embodiment the tumor cell is resistant to the apoptoticchemotherapeutic agent. In another embodiment the tumor cell issensitive to the apoptotic chemotherapeutic agent, and wherein theamount of metabolic modifying agent is effective to increasemitochondrial membrane potential and the amount of apoptoticchemotherapeutic agent is effective to inhibit the proliferation of thetumor cell when the mitochondrial membrane potential is increased.

According to yet another aspect of the invention a method for decreasingmitochondrial membrane potential in a cell of a subject is provided. Themethod includes the step of administering an MHC class II HLA-DR ligandto the subject to selectively engage MHC class II HLA-DR on the surfaceof the cell in an amount effective to decrease mitochondrial membranepotential in the mammalian cell. In one embodiment the method isperformed in vivo. In another embodiment the method is performed exvivo. In this aspect of the invention mammalian cells include but arenot limited to antigen presenting cells, T cells, and tumor cells.

In yet another aspect the invention is a method for increasingmitochondrial membrane potential in a mammalian cell expressing MHCclass II HLA-DP/DQ. The method includes the steps of administering anMHC class II HLA-DP/DQ ligand to the mammalian cell to selectivelyengage MHC class II HLA-DP/DQ on the surface of the cell in an amounteffective to increase mitochondrial membrane potential in the mammaliancell.

In one embodiment the mammalian cell is a pancreatic β cell of a type IIdiabetic and wherein the MHC class II HLA-DP/DQ ligand is administeredto the pancreatic β cell in vivo.

According to another aspect the invention is a method for decreasingmitochondrial membrane potential in a mammalian cell expressing MHCclass II HLA-DR. The method involves the steps of administering an MHCclass II HLA-DR ligand to the mammalian cell to selectively engage MHCclass II HLA-DR on the surface of the cell in an amount effective todecrease mitochondrial membrane potential in the mammalian cell.Preferably the mammalian cell is a pancreatic β cell of a type Idiabetic and wherein the MHC class II HLA-DR ligand is administered tothe pancreatic β cell in vivo. In one embodiment the mammalian cell is atumor cell and wherein the MHC class II HLA-DR ligand is administered tothe tumor cell in vivo.

The invention in another aspect is a method for treating a subjecthaving a tumor sensitive to treatment with a combination of an apoptoticchemotherapeutic agent and a metabolic modifying agent. The methodincludes the steps of administering to a subject in need of suchtreatment an apoptotic chemotherapeutic agent and a metabolic modifyingagent in a combined amount effective to inhibit growth of the tumor,said combined amount being an amount of apoptotic chemotherapeutic agentand an amount of metabolic modifying agent, wherein the amount ofmetabolic modifying agent is effective to increase mitochondrialmembrane potential and the amount of apoptotic chemotherapeutic agent iseffective to inhibit the proliferation of the tumor cell when themitochondrial membrane potential is increased.

According to another aspect the invention is a method for treating asubject having a tumor that is resistant to chemotherapy. The methodincludes the steps of administering to the subject an amount of anapoptotic chemotherapeutic agent, and administering substantiallysimultaneously therewith an amount of a metabolic modifying agent,wherein said amounts when administered are effective for inhibitinggrowth of the tumor.

According to another aspect the invention is a method for inducing theexpression of immune recognition molecules on a cell surface. The methodinvolves the step of contacting a cell with an amount of a metabolicinhibition agent effective to decrease mitochondrial membrane potential,wherein a decrease in mitochondrial membrane potential causes inductionof the expression of immune recognition molecules on the cell surface.Preferably the immune recognition molecule is selected from the groupconsisting of MHC class II, B7-1, B7-2, and CD-40. Preferably themetabolic inhibition agent is selected from the group consisting ofapoptotic chemotherapeutic agents, bacterial byproducts, mycobacterialantigens, UCP expression vectors, and fatty acids.

The invention in another aspect is a method for inhibiting pancreatic βcell death in a Type I diabetic. The progression of pancreatic β celldeath in type I diabetes involves two steps. The first phase of type Idiabetes is the insulitis phase which results when membrane potential isincreased, the β cells become cell surface Fas positive, but Fas-deathinsensitive. During this stage it is desirable to decrease the membranepotential and cause the cells to use fatty acids for fuel and becomecell surface Fas negative. If the diabetes is not treated during thefirst phase then it progresses to a second phase. During the secondphase the membrane potential is decreased and the β cell is induced todie if it remains cell surface Fas positive. Thus the inventioncontemplates a two phase approach to the treatment of type I diabetes.In the first phase a subject is treated to decrease the membranepotential of the pancreatic β cells to prevent or reduce the chance thatthe disease will progress from the insulitis phase to the cell deathphase. In the case when the disease has already progressed to the celldeath phase a subject is treated to increase the membrane potential oftheir pancreatic β cells. This method involves the steps of contacting apancreatic β cell of a Type I diabetic with an amount of a metabolicmodifying agent effective to increase mitochondrial membrane potentialin the pancreatic β cell. Preferably the metabolic modifying agent isselected from the group consisting of glucose, phorbol myristate acetatein combination with ionomycin, MHC class II HLA-DP/DQ ligand, GDP, CD40binding peptide, sodium acetate, UCP antisense, dominant negative UCP,and staurosporine. The method is also useful for promoting wound healingin a diabetic. In one embodiment the metabolic modifying agent isinfused with an antagonist of glucose, 2 deoxyglucose.

According to another aspect of the invention a method for inhibitingpancreatic β cell death in a Type I diabetic is provided. The methodinvolves the step of contacting a pancreatic p cell of a Type I diabeticwith an amount of a Fas binding agent effective to inhibit selectiveengagement of Fas on the surface of the pancreatic β cell.

According to yet another aspect of the invention a method for inducingpancreatic β cell death in a Type II diabetic is provided. The methodincludes the steps of contacting a pancreatic p cell of a Type IIdiabetic with an amount of an MHC class II HLA-DR inducing agenteffective to induce the expression of the MHC class II HLA-DR on thesurface of the pancreatic β cell, and selectively engaging the MHC classII HLA-DR by contacting the cell with an MHC class II HLA-DR ligandeffective to induce pancreatic β cell death. The MHC class II HLA-DRinducing agent is selected from the group consisting of adriamycin,gamma interferon, bacterial byproducts such as lipopolysaccharides,mycobacterial antigens such as BCG, a UCP expression vector, a TCRαβengagement molecule and a fatty acid in one embodiment.

In another aspect the invention is a method for treating a subjecthaving autoimmune disease to reduce associated cell death. The methodincludes the step of administering an amount of a γδ binding peptideeffective to specifically bind to and inactivate γδ cells in thesubject, wherein the inactivation of the γδ cells inhibits cell deathassociated with autoimmune disease. Preferably the γδ binding peptide isan anti-γδ antibody.

According to another aspect of the invention a method for treating asubject having autoimmune disease to reduce associated cell death isprovided. The method includes the steps of providing an extracellularenvironment having a high concentration of glucose to stimulateinduction of MHC class II HLA-DP/DQ and a low concentration of fattyacids to inhibit induction of MHC class II HLA-DR, wherein surfaceexpression of MHC class II HLA-DP/DQ is indicative of reduced cell deathassociated with autoimmune disease.

A method for screening a subject for susceptibility to atherosclerosisis provide according to another aspect of the invention. The methodincludes the steps of isolating a cell selected from the groupconsisting of peripheral blood lymphocyte and skin from a subject anddetecting the presence of an MHC marker selected from the groupconsisting of an MHC class II HLA-DP/DQ and MHC class II HLA-DR on thesurface of the cell selected from the group consisting of peripheralblood lymphocyte and skin, wherein the presence of MHC class IIHLA-DP/DQ is indicative of susceptibility to atherosclerosis and thepresence of MHC class II HLA-DR is indicative of resistance toatherosclerosis.

The invention in another aspect is a method for selectively killing aFas ligand bearing tumor cell. The method includes the step ofcontacting the a Fas ligand bearing tumor cell with acetate in an amounteffective to induce Fas associated cell death. In one embodiment the aFas ligand bearing tumor cell is contacted with the acetate in an amounteffective to sensitize the cell to a chemotherapeutic agent and furthercomprising the step of contacting the cell with a chemotherapeuticagent. A preferred chemotherapeutic agent is methotrexate. The methodmay also involve the step of administering a Fas ligand to the a Fasligand bearing tumor cell. In a preferred embodiment the Fas ligandbearing tumor cell is selected from the group consisting of a melanomacell and a colon carcinoma cell.

In another aspect the invention is a method for promoting a Th1 immuneresponse. The method involves the step of administering to a subject whohas been exposed to an antigen an effective amount for inducing a Th1immune response of a MHC class II HLA-DR inducing agent to induce DR ona T cell. In one embodiment the MHC class II HLA-DR inducing agent isfatty acid.

The invention also includes screening assays. A method for screening atumor cell of a subject for susceptibility to treatment with achemotherapeutic agent, is one aspect of the invention. The assayincludes at least the following steps: isolating a tumor cell from asubject; exposing the tumor cell to a chemotherapeutic agent; and,detecting the presence of a cell death marker selected from the groupconsisting of a Fas molecule on the surface of the tumor cell, a B7molecule on the surface of the tumor cell, an MHC class II HLA-DR on thesurface of the tumor cell, and a mitochondrial membrane potentialindicative of cellular coupling wherein the presence of the cell deathmarker indicates that the cell is susceptible to treatment with achemotherapeutic agent.

In one embodiment the cell death marker is a Fas molecule on the surfaceof the tumor cell and wherein the method comprises the step ofcontacting the Fas molecule with a detection reagent that selectivelybinds to the Fas molecule to detect the presence of the Fas molecule. Inanother embodiment the cell death marker is a MHC class II HLA-DRmolecule on the surface of the tumor cell and wherein the methodcomprises the step of contacting the MHC class II HLA-DR molecule with adetection reagent that selectively binds to the MHC class II HLA-DRmolecule to detect the presence of the MHC class II HLA-DR molecule.

Another screening assay of the invention is a method for identifying ananti-tumor drug for killing a tumor cell of a subject and includes thesteps of isolating a tumor cell from a subject; detecting the presenceof a cell death marker selected from the group consisting of a Fasmolecule on the surface of the tumor cell, a B7 molecule on the surfaceof the tumor cell, an MHC class II HLA-DR on the surface of the tumorcell, and a mitochondrial membrane potential indicative of cellularcoupling; exposing the tumor cell to a putative drug; and, detecting anychange in the presence of the cell death marker to determine whether theputative drug is an anti-tumor drug capable of killing the tumor cell ofthe subject.

a plurality of tumor cells is isolated from the subject and theplurality of tumor cells is screened with a panel of putative drugs inone embodiment of the assay. In another embodiment the change in thepresence of the cell death marker is detected by contacting the tumorcell with a cell death ligand attached to a solid support. Preferablythe cell death ligand is a Fas ligand.

Yet another assay of the invention is a method for screening a subjectfor susceptibility to disease. This method involves the steps ofisolating a cell selected from the group consisting of peripheral bloodlymphocyte and skin from a subject; and, detecting the presence of anMHC marker selected from the group consisting of an MHC class IIHLA-DP/DQ, B7-2, B7-1 and MHC class II HLA-DR on the surface of thecell, wherein the presence of MHC class II HLA-DP/DQ is indicative ofsusceptibility to atherosclerosis and resistance to autoimmune diseaseand the presence of MHC class II HLA-DR, B7-2, or B7-1 is indicative ofresistance to atherosclerosis and susceptibility to autoimmune disease.

The invention also encompasses kits. One kit of the invention is a kitfor screening a subject for susceptibility to disease. The kit includesa container housing a first binding compound that selectively binds to aprotein selected from the group consisting of B7-2, B7-1 and MHC classII HLA-DR; a container housing a second binding compound thatselectively binds to a MHC class II HLA-DP/DQ protein; and instructionsfor determining whether an isolated cell of a subject selectivelyinteracts with the first or second binding compound, wherein thepresence of MHC class II HLA-DP/DQ on the cell surface which interactswith the second compound is indicative of susceptibility toatherosclerosis and resistance to autoimmune disease and the presence ofMHC class II HLA-DR on the cell surface which interacts with the firstcompound is indicative of resistance to atherosclerosis andsusceptibility to autoimmune disease.

Another kit of the invention is a kit for screening a tumor cell of asubject for susceptibility to treatment with a chemotherapeutic agent.The kit includes a container housing a cell death marker detectionreagent; and instructions for using the cell death marker detectionreagent for detecting the presence of a cell death marker selected fromthe group consisting of a Fas molecule on the surface of the tumor cell,an MHC class II HLA-DR on the surface of the tumor cell, and amitochondrial membrane potential indicative of cellular coupling whereinthe presence of the cell death marker indicates that the cell issusceptible to treatment with a chemotherapeutic agent.

In some embodiments the kit also includes a container housing achemotherapeutic agent or a panel of chemotherapeutic agents, housed inseparate compartments. In other embodiments the kit also includes a celldeath ligand. Preferably the cell death ligand is coated on a solidsurface. In another preferred embodiment the cell death ligand is a Fasligand.

The invention in another aspect is a method for selectively killing acell. The method involves the step of contacting the cell with a nucleicacid selected form the group consisting of a UCP anti-sense nucleic acidand a UCP dominant-negative nucleic acid in an amount effect to inhibitUCP function.

In one embodiment the cell death marker is a Fas molecule on the surfaceof the tumor cell and wherein the method comprises the step ofcontacting the Fas molecule with a detection reagent that selectivelybinds to the Fas molecule to detect the presence of the Fas molecule. Inanother embodiment the cell death marker is a MHC class II HLA-DRmolecule on the surface of the tumor cell and wherein the methodcomprises the step of contacting the MHC class II HLA-DR molecule with adetection reagent that selectively binds to the MHC class II HLA-DRmolecule to detect the presence of the MHC class II HLA-DR molecule.

In another aspect the invention is a composition of a metabolicmodifying agent and an apoptotic chemotherapeutic agent. Preferably themetabolic modifying agent is selected from the group consisting ofglucose, phorbol myristate acetate in combination with ionomycin, MHCclass II HLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate,UCP antisense, dominant negative UCP, and staurosporine. In a preferredembodiment the apoptotic chemotherapeutic agent is selected from thegroup consisting of adriamycin, cytarabine, doxorubicin, andmethotrexate.

In one embodiment the metabolic modifying agent and the apoptoticchemotherapeutic agent are present in an amount effective to inhibit theproliferation of a tumor cell. In another embodiment the compositionincludes a pharmaceutically acceptable carrier.

The invention according to another aspect is a composition of an MHCclass II HLA-DR inducing agent and an MHC class II HLA-DR ligand. In oneembodiment the MHC class II HLA-DR inducing agent is selected from thegroup consisting of adriamycin, gamma interferon, bacterial byproductssuch as lipopolysaccharides, mycobacterial antigens such as BCG, a UCPexpression vector, a TCRαβ engagement molecule and a fatty acid. Inanother embodiment the MHC class II HLA-DR ligand is selected from thegroup consisting of an anti-MHC class II HLA-DR antibody, CD4 molecules,αβ T cell receptor molecules, γδ cell receptor molecules and a MHC classII HLA-DR binding peptide. According to yet another embodiment the MHCclass II HLA-DR inducing agent and the MHC class II HLA-DR ligand arepresent in an amount effective to lyse a tumor cell. The composition maybe formulated in a pharmaceutically acceptable carrier.

The invention also includes the discovery that neural differentiationand apoptosis are regulated through interaction of the immunerecognition molecules on the nerve cell surface with an NGF producingcell that expresses the counterpart surface immune recognition molecule,likely CD28 or CTLA4. The interaction between the nerve cell and the NGFproducing cell causes the NGF producing cell to release NGF into thelocal environment. This NGF then stimulates the nerve cell to undergonerve cell differentiation and innervation.

The invention in other aspects relates to methods and products forregulating nerve cell growth, differentiation, and apoptosis. In oneaspect the invention is a method for inducing nerve celldifferentiation. The method includes the steps of contacting a nervecell with an amount of a B7 inducing agent effective to induce theexpression of B7 on the surface of the nerve cell, and exposing thenerve cell to a neural activating cell to cause differentiation of thenerve cell.

In another aspect the invention is a method for inducing nerve celldifferentiation. The method involves the step of contacting a nerve cellwith an amount of a B7 inducing agent effective to induce the expressionof B7 on the surface of the nerve cell in the presence of an endogenousneural activating cell.

In some embodiments the B7 inducing agent is adriamycin, gammainterferon, a fatty acid, a lipoprotein, an anti-MHC class II HLA-DRantibody, a MHC class II HLA-DR binding peptide, a B7 expression vector,or a UCP expression vector.

In another embodiment the method also includes the step of contactingthe nerve cell with an amount of a metabolic modifying agent, which whenexposed to a cell causes increased coupling of electron transport andoxidative phosphorylation, effective to prevent dissipation of protonmotor force in the nerve cell prior to contacting the nerve cell withthe B7 inducing agent. In some embodiments the metabolic modifying agentis glucose, phorbol myristate acetate in combination with ionomycin, MHCclass II HLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate,UCP antisense, dominant negative UCP, and staurosporine. In otherembodiments the neural activating cell is a T cell, a macrophage, or adendritic cell.

In yet another embodiment the method includes the step of administeringa fatty acid to the nerve cell to stop cell division.

In yet another embodiment the method includes the step of inducing theexpression of a receptor for nerve growth factor.

According to another aspect the invention is a method for inducingapoptosis in a nerve cell. The method includes the steps of contacting anerve cell with an amount of a metabolic modifying agent, which whenexposed to a nerve cell causes an increase in coupling of electrontransport and oxidative phosphorylation, effective to preventdissipation of proton motor force in the nerve cell, and contacting aneural activating cell with an amount of a B7 receptor blocking agenteffective for inducing apoptosis in the nerve cell.

The metabolic modifying agent, in various embodiments, is glucose,phorbol myristate acetate in combination with ionomycin, MHC class IIHLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate, UCPantisense, dominant negative UCP, or staurosporine. In various otherembodiments the B7 receptor blocking agent is an anti-CD28 antibody,CD28 binding peptide, CTLA4 analog, anti-CTLA4 antibody, or CTLA4binding peptide.

The invention also includes compositions related to the above methods.In one aspect the invention is a composition of a metabolic modifyingagent and a B7 receptor blocking agent.

The metabolic modifying agent, in various embodiments, is glucose,phorbol myristate acetate in combination with ionomycin, MHC class IIHLA-DP/DQ ligand, GDP, CD40 binding peptide, sodium acetate, UCPantisense, dominant negative UCP, or staurosporine. In various otherembodiments the B7 receptor blocking agent is an anti-CD28 antibody,CD28 binding peptide, CTLA4 analog, anti-CTLA4 antibody, or CTLA4binding peptide.

In another embodiment the metabolic modifying agent and the B7 receptorblocking agent are present in an amount effective to induce apoptosis ofa nerve cell. In yet another embodiment the composition also includes apharmaceutically acceptable carrier.

A composition of a B7 inducing agent and a CD28 inducing agent isprovided in another aspect of the invention.

In some embodiments the B7 inducing agent is adriamycin, gammainterferon, bacterial byproducts such as lipopolysaccharides andlipoproteins, mycobacterial antigens such as BCG, and fatty acids, ananti-MHC class U HLA-DR antibody, a MHC class II HLA-DR binding peptide,a B7 expression vector, or a UCP expression vector. In other embodimentsthe CD28 inducing agent is a T cell receptor engagement molecule, CD3engagement molecule, IL4, or a CD28 expression vector. In yet anotherembodiment the composition also includes a pharmaceutically acceptablecarrier.

According to another aspect of the invention a method for re-innervatingan injured tissue is provided. The method includes the step ofimplanting a B7 expressing nerve cell in the injured tissue, wherein theimplanted B7 expressing nerve cell will undergo neuronal differentiationin the presence of a neural activating cell in the injured tissue tore-innervate the injured tissue.

In one embodiment the B7 expressing nerve cell constitutively expressesB7. In another embodiment the B7 expressing nerve cell is a nerve cellwhich constitutively expresses a UCP gene. In yet another embodiment theB7 expressing nerve cell is a nerve cell which constitutively expressesa B7 gene.

The method in another embodiment includes the step of administering a B7inducing agent effective to induce endogenous B7 expression on thesurface of the nerve cell.

The injured tissue may be any tissue in which a nerve is damaged. In oneembodiment the injured tissue is a spinal chord. In another embodimentthe injured tissue is a severed limb.

A method for treating a neurodegenerative disorder is provided accordingto another aspect of the invention. The method includes the step ofadministering an amount of a B7 inducing agent effective to induce theexpression of B7 on the surface of a nerve cell.

In some embodiments the B7 inducing agent is adriamycin, gammainterferon, bacterial byproducts such as lipopolysaccharides andlipoproteins, mycobacterial antigens such as BCG, and fatty acids, ananti-MHC class II HLA-DR antibody, a MHC class II HLA-DR bindingpeptide, a B7 expression vector, or a UCP expression vector.

The method may also include the step of inducing expression of CD28 onthe surface of a neural activating cell. Preferably the neuralactivating cell is a T cell. In other preferred embodiments the neuralactivating cell is a macrophage, a B cell or a dendritic cell.

In yet another embodiment the neurodegenerative disorder is selectedfrom the group consisting of paralysis, Parkinson's disease, Alzheimer'sdisease, amyotrophic lateral sclerosis, and multiple sclerosis.

According to another aspect the invention is a method for selectivelykilling a cell. The method includes the step of contacting the cell witha nucleic acid selected form the group consisting of a UCP anti-sensenucleic acid and a UCP dominant-negative nucleic acid in an amounteffect to inhibit UCP function.

In other aspects the invention is a method for selectively killing atumor cell. The method includes the steps of contacting the tumor cellwith acetate in an amount effective to induce cell surface Fasexpression, and administering a Fas ligand to the tumor cell in anamount effective to induce Fas associated cell death. In one embodimentthe tumor cell is contacted with the acetate in an amount effective tosensitize the cell to a chemotherapeutic agent and further comprisingthe step of contacting the cell with an apoptopic chemotherapeuticagent.

A method for selectively killing a tumor cell is provided according toanother aspect of the invention. The method includes the step ofcontacting the tumor cell with a compound selected from the groupconsisting of acetate, GDP and an apoptopic chemotherapeutic agent in anamount effective to kill the tumor cell.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each method and product.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more easily and completely understood whentaken in conjunction with the accompanying figures.

FIG. 1 shows a flow cytometric analysis of Fas expression.

FIG. 2 shows Intracellular and surface Fas Expression on L1210, upperpanels, and L1210/DDP cells, lower panels.

FIG. 3. Shows that treatment of L1210 DDP cells with staurosporinrestores Fas expression and susceptibility to drug-induced apoptosis.

FIG. 4 is a graph showing wild type and/or drug resistant cells expressintracellular and surface Fas.

FIG. 5. Is a graph showing glucose utilization and oxidation in normalrat islets.

FIG. 6 is a graph showing that glucose concentration, the large β cellsubset of gated cells have increased Fas expression and concomitantincreased mitochondrial membrane potential, while the smaller (possiblyalpha, glucagon producing cells) do not.

FIG. 7 shows mitochondrial membrane potential, assessed flowcytometrically using mitotracker red.

FIG. 8 shows a comparative analysis of alterations in levels of cAMP.

FIG. 9 shows an analysis of DNA fragmentation from resting B cells.

FIG. 10 shoes that treatment of resting B lymphocytes with anti-class IImAb results in B cell apoptosis, as measured by increases innucleosome-sized DNA fragments.

FIG. 11 shows a two-color flow cytometric analysis of apoptotic restingB-cells.

FIG. 12A demonstrates that in contrast to B-cells from normal mice,resting B-cells from (NZB×SWR)F1 and (NZB×NZW)F1 mice do not elevateintracellular cAMP in response to ligation of their class II molecules.

FIG. 12B shows a ligation of class II molecules on resting B-cells from(NZB×NZW)F1 and (NZB×SWR)F1 animals does not result in apoptotic death.

FIG. 13 shows UCP expression in L1210 and L1210/DDP cells in response tostaurosporin and PMA.

FIG. 14 demonstrates that UCP is expressed in a panel of Tumor Cells.

FIG. 15 shows that flow cytometrically detected UCP expression wasmitochondrial using isolated mitochondria from L1210 and L1210 DDP, andWestern Blot analysis blotting with rabbit anti-UCP antibodies.

FIG. 16 shows that increased UCP corresponds to increased mitochondrialproton leak and a lower mitochondrial membrane potential (ΔQm).

FIG. 17 depicts the level of cell surface Fas expression onnon-permeabilized (panel A) and intracellular Fas expression inpermeabilized (Panel B) B16 melanoma cells.

FIG. 18 depicts the rates of glucose utilization and oxidation in B16melanoma cells.

FIG. 19 shows that possibility that normal mouse T cells express E.

FIG. 21 shows the results of fatty acid (Oleic Acid) as a mitochondrialcarbon source.

FIG. 22 shows levels of cAMP in L1210, left panel versus L1210DDP, rightpanel.

FIG. 23 is a graph depicting Sodium Acetate as a mitochondrial modifyingagent.

FIG. 24 is a graph depicting the effects of acetate on susceptibility toFas-dependent cell death.

FIG. 25 is a histogram depicting the constitutive expression or lackthereof of cell surface molecules Fas (panels A, D, G), B7.1 (panels B,E H) and B7.2 (panels C, F, I) in PC12 cells (panels A, B, C), PC12/TrkAcells (Panels D, E, F) and PC12/v-Crk Cells (panels G, H, I).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the human B7 (B7.1) cDNA withGenBank Acc. no.: M27533.

SEQ ID NO:2 is the predicted amino acid sequence of the translationproduct of human B7 (B7.1) cDNA (SEQ ID NO:1).

SEQ ID NO:3 is the nucleotide sequence of the human B7.2 cDNA withGenBank Acc. no. U04343.

SEQ ID NO:4 is the predicted amino acid sequence of the translationproduct of human B7.2 cDNA (SEQ ID NO:3).

SEQ ID NO:5 is the nucleotide sequence of the human uncoupling (UCP-1)cDNA with GenBank Acc. no. U28480.

SEQ ID NO:6 is the predicted amino acid sequence of the translationproduct of human uncoupling cDNA (UCP-1) (SEQ ID NO:5).

SEQ ID NO:7 is the nucleotide sequence of the human uncoupling (UCP-2)cDNA with GenBank Acc. no. U82819.

SEQ ID NO:8 is the predicted amino acid sequence of the translationproduct of human uncoupling cDNA (UCP-2) (SEQ ID NO:7).

SEQ ID NO:9 is the nucleotide sequence of the human uncoupling (UCP-3S)cDNA with GenBank Acc. no. U82818.

SEQ ID NO:10 is the predicted amino acid sequence of the translationproduct of human uncoupling cDNA (UCP-3S) (SEQ ID NO:9).

SEQ ID NO:11 is the nucleotide sequence of the human CD28 cDNA withGenBank Acc. no. J02988.

SEQ ID NO:12 is the predicted amino acid sequence of the translationproduct of the human CD28 cDNA (SEQ ID NO:11).

SEQ ID NO:13 is the amino acid sequence of a peptide.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and products involving the control ofcell division, differentiation, death, and apoptosis by the regulationof cell surface immune recognition molecules. It was discoveredaccording to one aspect of the invention that proton motor force(assessed as mitochondrial metabolism) is integrally related to theregulation of cellular division and cellular apoptosis. The ability tomanipulate mitochondrial metabolic processes has led to the developmentof methods for treating diseases associated with excessive cellularproliferation or premature cellular death. Additionally, the ability tomanipulate the expression of cell surface immune recognition moleculessuch that a nerve cell can stimulate local NGF production from an NGFproducing cell has led to the development of methods for treatingneurodegenerative diseases associated with premature cellular death. Itwas also discovered according to the invention that the regulation ofproton motor force (mitochondrial metabolism) is directly related to theexpression of these cell surface immune recognition molecules involvedin the signaling process of cell death and in immune response signaling,and thus can be manipulated as one method for regulating the expressionof the immune recognition molecules. The ability to control theexpression of these cell surface molecules is a useful and powerfultechnique for therapeutically manipulating the processes of cellulardeath, apoptosis, differentiation and proliferation. Monitoring theexpression of these proteins is also useful for screening assays toassess disease states as well as the mitochondrial metabolic status ofcells.

Based on all these discoveries the invention includes in some aspectsmethods for increasing or decreasing the mitochondrial membranepotential in a mammalian cell. The ability to manipulate themitochondrial membrane potential of a cell provides the ability tocontrol the fate of the cell. When the membrane potential of a cell isdecreased and the cell is caused to use potential of a cell isincreased, however, and the cell is using glucose for fuel, the samesignal can be interpreted as a signal to divide rather than for celldeath. The invention encompasses mechanisms for controlling thesecomplex interactions to regulate the processes of cellular death anddivision.

One method for causing a decrease in mitochondrial membrane potentialand a switch to the use of fatty acids as fuel is by inducing theexpression of MHC class II HLA-DR on the surface of the cell. If lowamounts of MHC class II HLA-DR are already expressed on the surface thecell can be contacted with an MHC class II HLA-DR ligand to cause afurther decrease in the mitochondrial membrane potential. When a cellhas been induced to express MHC class II HLA-DR on the cell surface suchthat the electron transport is uncoupled and the cell is using fattyacids for fuel and the cell is contacted with a MHC class II HLA-DRligand, then the cell generally will interpret that signal as a celldeath signal, and cause cell lysis.

The invention also encompasses methods for causing an increase inmitochondrial membrane potential. This increase, accompanied by the useof glucose as fuel is accomplished in some aspects by inducing theexpression of MHC class II HLA-DPDQ on the surface of the cell. If lowamounts of MHC class II HLA-DPDQ are already expressed on the surfacethe cell can be contacted with an MHC class II HLA-DPDQ ligand to causea further increase in the mitochondrial membrane potential and anincrease in coupling of electron transport and oxidativephosphorylation. When a cell has been induced to express MHC class IIHLA-DPDQ on the cell surface such that the electron transport isrelatively coupled and the cell is using glucose for fuel and the cellis contacted with a MHC class II HLA-DPDQ ligand, then the cellgenerally will interpret that signal as a cell division signal, andcause cellular division.

The methods of the invention have broad utility in regulating mammaliancell growth and death in vitro, in vivo and ex vivo. Because mammaliancells utilize the basic process of mitochondrial metabolism inregulating their own growth and differentiation, any type of mammaliancell can be manipulated according to the methods of the invention. Whenthe methods for increasing or decreasing mitochondrial metabolism areperformed in vitro by contacting an MHC class II HLA-DPDQ or -DRexpressing cell with an MHC class II HLA-DPDQ or -DR ligand,respectively, the methods are not performed on antigen presenting cells.When the same methods are performed ex vivo or in vivo they may however,be performed on antigen presenting cells as well as any other type ofmammalian cell. An “antigen presenting cell” is used herein consistentlywith its well known meaning in the art and includes, for instance, isused herein consistently with its well known meaning in the art andincludes, for instance, dendritic cells, macrophage, etc. The in vitromethods of the invention are useful for a variety of purposes. Forinstance, the methods of the invention may be useful for identifyingdrugs which have an effect, such as a preventative effect, on cellulardivision or death by contacting cells which are caused by themanipulations of the invention to undergo cellular division or death.

In addition to the in vitro methods, the methods of the invention may beperformed in vivo or ex vivo in a subject to manipulate one or more celltypes within the subject. An “ex vivo” method as used herein is a methodwhich involves isolation of a cell from a subject, manipulation of thecell outside of the body, and reimplantation of the manipulated cellinto the subject. The ex vivo procedure may be used on autologous orheterologous cells, but is preferably used on autologous cells. Inpreferred embodiments, the ex vivo method is performed on cells that areisolated from bodily fluids such as peripheral blood or bone marrow, butmay be isolated from any source of cells. When returned to the subject,the manipulated cell will be programmed for cell death or division,depending on the treatment to which it was exposed. Ex vivo manipulationof cells has been described in several references in the art, includingEngleman, E. G., 1997, Cytotechnology, 25: 1; Van Schooten, W., et al.,1997, Molecular Medicine Today, June, 255; Steinman, R. M., 1996,Experimental Hematology, 24, 849; and Gluckman, J. C., 1997, Cytokines,Cellular and Molecular Therapy, 3:187. The ex vivo activation of cellsof the invention may be performed by routine ex vivo manipulation stepsknown in the art. In vivo methods are also well known in the art. Asubject as used herein means humans, primates, horses, cows, pigs,sheep, goats, dogs, cats and rodents. The invention thus is useful fortherapeutic purposes and also is useful for research purposes such astesting in animal or in vitro models of medical, physiological ormetabolic pathways or conditions.

In preferred embodiments of the invention the mammalian cell is a tumorcell The method is useful for inducing cell lysis in many types ofmammalian cells but is particularly useful for inducing cell lysis in atumor cell. A “tumor cell” as used herein is a cell which is undergoingunwanted mitotic proliferation. A tumor cell when used in the in vitroaspects of the invention can be isolated from a tumor within a subjector may be part of an established cell line. A tumor cell in a subjectmay be part of any type of cancer. Cancers include but are not limitedto biliary tract cancer; brain cancer, including glioblastomas andmedulloblastomas; breast cancer; cervical cancer; choriocarcinoma; coloncancer; endometrial cancer; esophageal cancer; gastric cancer;hematological neoplasms, including acute lymphocytic and myelogenousleukemia; multiple myeloma; AIDS associated leukemias and adult T-cellleukemia lymphoma; intraepithelial neoplasms, including Bowen's diseaseand Paget's disease; liver cancer; lung cancer; lymphomas, includingHodgkin's disease and lymphocytic lymphomas; neuroblastomas; oralcancer, including squamous cell carcinoma; ovarian cancer, includingthose arising from epithelial cells, stromal cells, germ cells andmesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor.

In the aspects of the invention that the mammalian cell is a tumor celland the cell is only treated with an MHC class II HLA-DR inducing agentbut not an MHC class II HLA-DR ligand the MHC class II HLA-DR inducingagent does not include adriamycin and gamma interferon. When the MHCclass II HLA-DR inducing agent is adriamycin or gamma interferon themethod of lysing the tumor cell requires the additional step ofcontacting the tumor cell with an MHC class II HLA-DR ligand to causecell lysis.

Cell lysis is the necrotic death of a cell which occurs by osmoticrupture.

As used herein an “MHC class II HLA-DR inducing agent” is an agent whichcauses MHC class II HLA-DR to be expressed on the cell surface.Preferably the MHC class II HLA-DR inducing agent is a pharmacologicalagent that causes uncoupling of electron transport and oxidativephosphorylation, resulting in reduced mitochondrial-membrane potentialwithin the cell. MHC class II HLA-DR inducing agents include but are notlimited to adriamycin, gamma interferon, bacterial byproducts such aslipopolysaccharides, mycobacterial antigens such as BCG, a UCPexpression vector, a TCRαβ engagement molecule and a fatty acid.Although gamma interferon induces expression of both MHC class II HLA-DRand MHC class II HLA-DP/DQ it can still be used in combination with anMHC class II HLA-DR ligand which selectively binds to MHC class IIHLA-DR and not MHC class II HLA-DP/DQ. The MHC class II HLA-DR inducingagent is an isolated molecule. An isolated molecule is one which hasbeen removed from its natural surroundings and formulated foradministration to an organism. Adriamycin, gamma interferon, bacterialbyproducts such as lipopolysaccharides, mycobacterial antigens such asBCG are all well known compounds which can be purchased from a varietyof commercial sources. UCP expression vector can be prepared by methodswell known in the art, such methods are described in detail below. Fattyacids are also well known compounds that can be purchased commerciallyfrom many sources. Preferred fatty acids include but are not limited tooleic acid, palmitate, and myristic acid. A “TCRαβ engagement molecule”as used herein refers to any compound that can bind to and cause cellsurface crosslinking of CD4 and the αβT cell receptor (αβTCR). Suchcompounds are known in the art. For instance heterobifunctionalantibodies are capable of crosslinking CD4 and αβTCR by interacting withboth molecules on the surface of the cell. Other CD4/αβTCR bindingmolecules can be identified with routine experimentation and are alsoencompassed by the term TCRαβ engagement molecule. Routine screeningmethods for identifying such binding molecules are set forth below.

MHC class II HLA-DR refers to a subregion of the human majorhistocompatibility class II genetic locus. As used herein the “MHC classII HLA-DR” is the protein expressed on the surface of a cell whichcorresponds to the MHC class II HLA-DR genetic locus. Although the termHLA-DR refers to the human subclass of MHC, the invention is intended toencompass the corresponding subclass of MHC in other species, which havedifferent nomenclature, such as the IE region in the correspondingsubclass in the mouse.

As used herein an “MHC class II HLA-DR ligand” is a molecule which bindsto MHC class II HLA-DR and stimulates an MHC class II HLA-DR specificintracellular signal stimulating cell lysis. MHC class II HLA-DR ligandsare MHC class II HLA-DR binding peptides which cause cell surfacecrosslinking of MHC class II HLA-DR molecules. Such ligands are wellknown in the art and include but are not limited to anti-MHC class IIHLA-DR antibodies such as those commercially available from BectonDickinson and many other sources, CD4 peptides, γδ T cell receptor (TCR)peptides, αβ TCR peptides, and other binding peptides, optionally boundto a delivery vehicle such as a liposome. CD4 peptides, γδTCR peptides,and αβ TCR peptides are well known cell surface molecules. Thesepeptides can be used as a ligand in a soluble form or may be attached orconjugated to a carrier such as a liposome or particle (otherchemical/physical vectors useful for this purpose are discussed below).In addition to these known binding peptides other MHC class II HLA-DRbinding peptides can be identified with routine experimentation and arealso encompassed by the term MHC class II HLA-DR ligand. Routinescreening methods for identifying such binding molecules are set forthbelow.

Cell lysis can be assessed by any method known in the art for makingsuch measurements. For example cell lysis can be determined by directhistological analysis, comparison of intact cell numbers using a coultercounter, and flow cytometry. These methods are well known in the art andsome are described in more detail in the examples section below.

The “MHC class II HLA-DR ligand” as used herein is an isolated molecule.An isolated molecule is one which has been removed from its naturalsurroundings and formulated for administration to an organism.

The methods of the invention in some aspects may also be performed usingendogenous MHC class II HLA-DR ligand. An “endogenous MHC class IIHLA-DR ligand” is different than an “MHC class II HLA-DR ligand” usedabove which is an isolated composition. For instance the endogenous MHCclass II HLA-DR ligand may be a cell having a cell surface MHC class IIHLA-DR binding peptide. In this case the method would only include thestep of contacting a tumor cell with an amount of an MHC class II HLA-DRinducing agent effective to induce the expression of MHC class II HLA-DRon the surface of the tumor cell in the presence of an endogenous MHCclass II HLA-DR ligand.

When the endogenous MHC class II HLA-DR ligand is a cell having a cellsurface MHC class II HLA-DR binding peptide which is already present ininteractive proximity to the MHC class II HLA-DR, the cell does not haveto be manually brought into contact with the MHC class II HLA-DR.

Another aspect of the invention involves the induction of apoptosis in atumor cell rather than cell lysis. In both apoptosis and cell lysis thecell dies but the processes occur through different mechanisms and whenthe cell is in a different metabolic state. As described above, when themethods of the invention are performed to induce cell lysis in a tumorcell the cell is in an uncoupled state. When the methods of theinvention are performed to induce apoptosis the cell is caused to assumea coupled state. The method for inducing apoptosis in a tumor cellinvolves the steps of contacting a tumor cell with an amount of ametabolic modifying agent, which when exposed to a cell causes couplingof electron transport and oxidative phosphorylation, effective toincrease the mitochondrial membrane potential in the tumor cell, andcontacting the tumor cell with an amount of a chemotherapeutic agenteffective for inducing apoptosis in the tumor cell.

Apoptosis is a process of cell death in which the cell undergoesshrinkage and fragmentation, followed by phagocytosis of the cellfragments. Apoptosis is well known in the art and can be assessed by anyart recognized method. For example apoptosis is easily determined usingflow cytometry, which distinguishes between live and dead cells. Flowcytometry is described in more detail in the Examples below.

As used herein a “metabolic modifying agent” is an agent which whenexposed to a cell causes coupling of electron transport and oxidativephosphorylation, resulting in increased mitochondrial membrane potentialwithin the cell. Metabolic modifying agents include but are not limitedto glucose, sodium acetate, phorbol myristate acetate in combinationwith ionomycin, MHC class II HLA-DP/DQ ligand, guanosine diphosphate(GDP), CD40 binding peptide, sodium acetate, UCP antisense, dominantnegative UCP, and staurosporine. Glucose, phorbol myristate acetate,ionomycin, GDP, and staurosporine are all well known commerciallyavailable compounds which can be obtained form many sources. CD40binding peptides are any peptide molecules which interact with CD40,causing CD40 crosslinking on a cell surface. These molecules include,for example, CD40 ligand, which is a well known molecule. CD40 bindingpeptides are not limited to CD40 ligand, however, but include othermolecules which can be identified with routine experimentation. Routinescreening methods for identifying such binding molecules are set forthbelow. UCP antisense molecules and dominant negative UCP molecules arealso known in the art and are described in more detail below.

MHC class II HLA-DP/DQ refers to another subregion of the human majorhistocompatibility class II genetic locus. As used herein the “MHC classII HLA-DP/DQ” is the protein expressed on the surface of a cell whichcorresponds to the MHC class II HLA-DP/DQ genetic locus. Although theterm HLA-DP/DQ refers to the human subclass of MHC, the invention isintended to encompass the corresponding subclass of MHC in otherspecies, which have different nomenclature, such as the IA region in thesubclass in the mouse.

As used herein an “MHC class II HLA-DP/DQ ligand” is a molecule whichbinds to MHC class II HLA-DP/DQ and stimulates an MHC class II HLA-DP/DQspecific intracellular signal stimulating coupling of electron transportand oxidative phosphorylation resulting in increased mitochondrialmembrane potential. MHC class II HLA-DP/DQ ligands include but are notlimited to anti-MHC class II HLA-DP/DQ ligand antibodies, other bindingpeptides, and cells having a cell surface MHC class II HLA-DP/DQ bindingantigen. When the MHC class II HLA-DP/DQ ligand is a cell having a cellsurface MHC class II HLA-DP/DQ binding antigen which is already presentin interactive proximity to the MHC class II HLA-DP/DQ, the cell doesnot have to be manually brought into contact with the MHC class IIHLA-DP/DQ.

As used herein, the term “dissipation of proton motor force” refers tothe relative amount of protons in the mitochondria. It can be assessedby measuring mitochondrial membrane potential. As used herein“mitochondrial membrane potential” is the pressure on the inside of themitochondrial cell membrane measured relative to the extracellular fluidwhich is created by the generation and dissipation of charge within themitochondria. The mitochondrial membrane potential is maintained by theenergy generating system of the mitochondria. In most tissues electrontransport is coupled to oxidative phosphorylation resulting in theproduction of ATP from glucose. Uncoupling proteins (UCPs) can cause thereversible uncoupling of electron transport and oxidativephosphorylation, which leads to a decrease in the mitochondrial membranepotential. Other tissue, often referred to as the immuno-privilegedtissue such as the brain, testis, ovary, eye, and pancreatic β cells,express UCPs which cause electron transport to be uncoupled to oxidativephosphorylation under normal conditions. In these tissues glucose cannotbe converted to ATP while the UCP is active because of the uncouplingand the energy produced is converted into other energy forms such asheat and released. If the metabolic processing systems in these tissuesare caused to undergo coupling the membrane potential would increase.

The absolute levels of the mitochondrial membrane potential varydepending on the cell or tissue type. As used herein an “increase inmitochondrial membrane potential” is an increase relative to the normalstatus of the cell being examined and results from the prevention ofdissipation of proton motor force. “Prevention” as used herein refers toa decrease or reduction in the amount of dissipation that wouldordinarily occur in the absence of the stimulus applied according to themethods of the invention to cause coupling. If electron transport andoxidative phosphorylation are normally uncoupled within the cell thenthe baseline potential will be relatively low and when the ATPgenerating systems are coupled an increase in mitochondrial membranepotential from that baseline level is observed. Likewise, a “decrease inmitochondrial membrane potential” is a decrease relative to the normalstatus of the cell being examined and results from the dissipation ofproton motor force. If electron transport and oxidative phosphorylationare normally coupled within the cell then the baseline potential will berelatively high and when the ATP generating systems are uncoupled adecrease in mitochondrial membrane potential from that baseline level isobserved.

Changes in mitochondrial membrane potential can be assessed by anymethod known in the art for making such measurements. For example themitochondrial membrane potential may be measured cytometrically byincubating cells for 20 minutes at room temperature with 5 mg/ml JC-1³⁹a fluorescent probe able to bind mitochondria. The aggregation state andconsequently the fluorescence emission of JC-1 changes as themitochondrial membrane potential is altered. Valinomycin, whichcollapses the mitochondrial membrane potential can be used as a positivecontrol treatment. Flow cytometry permits the examination of up to fourfluorescent markers concurrently. This method is described in moredetail in the Examples section below In addition to examining themitochondrial membrane potential, studies can be performed to determinethe rate of glucose utilization and oxidation and measurements of protonleak can be assessed by a top-down elasticity analysis, each of which isdescribed in more detail in the Examples below.

The relationship between mitochondrial metabolism and cell surface Fasexpression is important to the methods of the invention. When a cell iscoupled Fas is expressed on the cell surface and when a cell isuncoupled Fas generally is transported to intracellular stores. When acell is coupled and Fas is on the surface engagement of Fas sends asignal to the cell instructing the cell to undergo cellular division. Ifa chemotherapeutic agent is added then the signal is changed to a signalwhich instructs the cell to undergo apoptosis. When a cell is uncoupledand ordinarily Fas is not expressed on the cell surface. Under certaindisease conditions such as Type I diabetes (discussed in more detailbelow), or when the cell has been irradiated Fas can be expressed on thesurface of uncoupled cells. When this occurs engagement of Fas sends asignal to the cell to die.

An “apoptotic chemotherapeutic agent” as used herein is a group ofmolecules which function by a variety of mechanisms to induce apoptosisin rapidly dividing cells. Apoptotic chemotherapeutic agents are a classof chemotherapeutic agents which are well known to those of skill in theart. Chemotherapeutic agents include those agents disclosed in Chapter52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and theintroduction thereto, 1202-1263, of Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Eighth Edition, 1990,McGraw-Hill, Inc (Health Professions Division), incorporated herein byreference. Suitable chemotherapeutic agents may have various mechanismsof action. The classes of suitable chemotherapeutic agents include (a)Alkylating Agents such as nitrogen mustard (e.g. mechlorethamine,cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines andmethylmelamines (e.g. hexamethylmelamine, thiotepa), alkyl sulfonates(e.g. busulfan), nitrosoureas (e.g. carmustine which is also known asBCNU, lomustine which is also known as CCNU semustine which is alsoknown as methyl-CCNU, chlorozoticin, streptozocin), and triazines (e.g.dicarbazine which is also known as DTIC); (b) Antimetabolites such asfolic acid analogs (e.g. methotrexate), pyrimidine analogs (e.g.5-fluorouracil floxuridine, cytarabine, and azauridine and its prodrugform azaribine), and purine analogs and related materials (e.g.6-mercaptopurine, 6-thioguanine, pentostatin); (c) Natural Products suchas the vinca alkaloids (e.g. vinblastine, Vincristine),epipodophylotoxins (e.g. etoposide, teniposide), antibiotics (e.g.dactinomycin which is also known as actinomycin-D, daunorubicin,doxorubicin, bleomycin, plicamycin, mitomycin, epirubicin, which is4-epidoxorubicin, idarubicin which is 4-dimethoxydaunorubicin, andmitoxanthrone), enzymes (e.g. L-asparaginase), and biological responsemodifiers (e.g. Interferon alfa); (d) Miscellaneous Agents such as theplatinum coordination complexes (e.g. cisplatin, carboplatin),substituted ureas (e.g. hydroxyurea), methylhydiazine derivatives (e.g.procarbazine), adreocortical suppressants (e.g. mitotane,aminoglutethimide) taxol; (e) Hormones and Antagonists such asadrenocorticosteroids (e.g. prednisone or the like), progestins (e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate), estrogens (e.g. diethyestilbestrol, ethinyl estradiol, and thelike), antiestrogens (e.g. tamoxifen), androgens (e.g. testosteronepropionate, fluoxymesterone, and the like), antiandrogens (e.g.flutamide), and gonadotropin-releasing hormone analogs (e.g. leuprolide)and (F) DNA damaging compounds such as adriamycin.

In addition to the methods of manipulating cells, the invention is alsouseful for screening cells such as tumor cells to determine if thosecells are susceptible to cellular division or cellular death, alone orin conjunction with treatment with a chemotherapeutic agent or othercell signal and kits for performing these screening assays. Thescreening method can be accomplished by isolating a tumor cell from asubject and exposing the tumor cell to a chemotherapeutic agent(preferably several different doses of several differentchemotherapeutic agents can be screened at a time). Then the presence ofa cell death marker can be detected. The level of the cell death markerindicates that the cell is susceptible to treatment with achemotherapeutic agent.

As used herein a “cell death marker” is a cell surface molecule whichindicates that the cell is susceptible to cell death. A variety of celldeath markers exist but the preferred cell death markers usefulaccording to the invention include a Fas molecule on the surface of thetumor cell, an MHC class II HLA-DR on the surface of the tumor cell, anda mitochondrial membrane potential indicative of cellular coupling. TheFas and MHC molecules can be detected by using a detection reagent thatbind to the protein, such as an antibody.

The screening methods are particularly useful for determining if a tumoris sensitive to a chemotherapeutic agent. A tumor, however, mayinitially be sensitive to a particular chemotherapeutic agent and thenas the therapy progresses the tumor may become resistant to thatchemotherapeutic agent. The methods of the invention can be used toprevent the tumor from becoming sensitive to a chemotherapeutic agentduring therapy. The method involves the steps of administering to asubject in need of such treatment a chemotherapeutic agent and ametabolic modifying agent in a combined amount effective to inhibitgrowth of the tumor. The metabolic modifying agent causes the electrontransport and oxidative phosphorylation processes to be coupled andtherefore effects an increased mitochondrial membrane potential in thecell. As the cell is held in this coupled state Fas is expressed on thesurface and the chemotherapeutic agent can stimulate Fas mediatedapoptosis. The cells will be prevented from becoming resistant.

The combined amount of metabolic modifying agent and apoptoticchemotherapeutic agent effective to inhibit growth of the tumor cell isthat amount is effective to inhibit the proliferation of the tumor cellwhen the mitochondrial membrane potential is increased. An effectiveamount means that amount necessary to delay the onset of, inhibit theprogression of, halt altogether the onset or progression of or diagnosethe particular condition being treated. In general, an effective amountfor treating a tumor cell is that amount necessary to halt theproliferation of the cell. In one embodiment, the effective amount isthat amount necessary to kill the cell. In general, an effective amountfor treating cancer will be that amount necessary to favorably affectmammalian cancer cell proliferation in-situ. When administered to asubject, effective amounts will depend, of course, on the particularcondition being treated; the severity of the condition; individualpatient parameters including age, physical condition, size and weight;concurrent treatment; frequency of treatment; and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

In some cases the screening assay may indicate that the tumor is mostlyresistant to a chemotherapeutic agent. Resistant tumors may also betreated by the methods of the invention. One aspect of the inventioninvolves the discovery that resistant tumors cells have a mitochondrialmetabolic state in which electron transport is uncoupled from oxidativephosphorylation. It was discovered according to the invention that byaltering the metabolic state of the tumor cell and thereby causingelectron transport to be coupled to oxidative phosphorylation it ispossible to cause the resistant cell to revert such that it becomessensitive to chemotherapy. The method is performed by administering tothe subject an amount of a chemotherapeutic agent, and substantiallysimultaneously therewith an amount of a metabolic modifying agent whichtogether are effective for inhibiting growth of the tumor. The metabolicmodifying agent causes electron transport in the cell to be coupled tooxidative phosphorylation. As discussed above once these processes arecoupled Fas is expressed on the surface and the cell becomes susceptibleto apoptosis induced by the chemotherapeutic agent.

Other screening assays can be performed according to the invention toidentify an anti-tumor drug for killing a tumor cell of a subject. Theseassays are accomplished by isolating a tumor cell from a subject;detecting the presence of a cell death marker selected from the groupconsisting of a Fas molecule on the surface of the tumor cell, a B7molecule on the surface of the tumor cell, an MHC class II HLA-DR on thesurface of the tumor cell, and a mitochondrial membrane potentialindicative of cellular coupling; exposing the tumor cell to a putativedrug; and, detecting any change in the presence of the cell death markerto determine whether the putative drug is an anti-tumor drug capable ofkilling the tumor cell of the subject. This assay may be performed onone or a plurality of tumor cells and with a single drug or with a panelof drugs.

The assay can be performed using routine equipment known in the art. Forinstance the change in the presence of the cell death marker can bedetected by contacting the tumor cell with a cell death ligand attachedto a solid support.

The invention also encompasses kits for screening a subject forsusceptibility to disease. This kit includes at least a containerhousing a first binding compound that selectively binds to a proteinselected from the group consisting of B7-2, B7-1 and MHC class IIHLA-DR; a container housing a second binding compound that selectivelybinds to a MHC class II HLA-DP/DQ protein; and instructions fordetermining whether an isolated cell of a subject selectively interactswith the first or second binding compound, wherein the presence of MHCclass II HLA-DP/DQ on the cell surface which interacts with the secondcompound is indicative of susceptibility to atherosclerosis andresistance to autoimmune disease and the presence of MHC class II HLA-DRon the cell surface which interacts with the first compound isindicative of resistance to atherosclerosis and susceptibility toautoimmune disease.

Other kits include kits for screening a tumor cell of a subject forsusceptibility to treatment with a chemotherapeutic agent. These kitsinclude a container housing a cell death marker detection reagent; andinstructions for using the cell death marker detection reagent fordetecting the presence of a cell death marker selected from the groupconsisting of a Fas molecule on the surface of the tumor cell, an MHCclass II HLA-DR on the surface of the tumor cell, and a mitochondrialmembrane potential indicative of cellular coupling wherein the presenceof the cell death marker indicates that the cell is susceptible totreatment with a chemotherapeutic agent. The kit may also include acontainer housing a chemotherapeutic agent. Optionally, the kit mayinclude a panel of chemotherapeutic agents, housed in separatecompartments.

The invention also involves the discovery that mitochondrial metabolicregulation is directly related to the expression of immune recognitionmolecules on a cell surface. As used herein “immune recognitionmolecules” are cell surface proteins which mark a cell foridentification by immune cells. Immune recognition molecules include butare not limited to MHC, and in particular MHC class II HLA-DR, B7-1,B7-2 and CD-40. When the mitochondrial metabolic status of the cell issuch that the electron transport is uncoupled to oxidativephosphorylation the cell surface expression of the immune recognitionmolecules is increased. When the mitochondrial metabolic status of thecell is such that the electron transport is coupled to oxidativephosphorylation the cell surface expression of the immune recognitionmolecules is decreased. Under these conditions, however, the expressionof MHC class II HLA-DP/DQ is actually increased. For purposes of thispatent application MHC class II HLA-DP/DQ is not defined as an immunerecognition molecule.

Based on these findings the invention encompasses a method for inducingthe expression of immune recognition molecules on a cell surface. Themethod involves contacting the cell with an amount of a metabolicinhibition agent effective to decrease mitochondrial membrane potential,wherein a decrease in mitochondrial membrane potential causes inductionof the expression of immune recognition molecules on the cell surface.

A “metabolic inhibition agent” as used herein is an agent that causeselectron transport to become uncoupled from oxidative phosphorylation,and includes for example apoptotic chemotherapeutic agents, bacterialbyproducts, mycobacterial antigens, UCP expression vectors, and fattyacids.

Diabetes mellitus, which encompasses both Type I (i.e., InsulinDependent Diabetes Mellitus (IDDM)) and Type II (i.e., Non-InsulinDependent Diabetes Mellitus (NIDDM)), is known to affect more than onehundred million individuals worldwide. Although the exact cause ofdiabetes is unclear it is believed that diabetes may arise from any of avariety of physiological conditions such as genetic syndromes, viralinfections, age related deterioration of structures responsible formaintaining the glycemic response, pancreatic disease, hormonalabnormalities, certain drugs or chemicals, insulin receptorabnormalities, etc. A “type I diabetic” is a subject who has diabetesmellitus caused by a destruction of beta cells in the pancreas. Type Idiabetics require daily insulin administration which may be reduced butnot altogether eliminated by careful restriction of diet.

Neither the genetic/environmental influences nor the inherent β cellcharacteristics that trigger immune-mediated destruction are completelyunderstood. However, two features that are pivotal in susceptibility toβ cell destruction are the expression of the cell surface molecule Fasand the metabolic state of the β cells. Fas can induce mitosis orapoptosis depending on the cell and the experimental circumstances.During the prediabetic stage of Type 1 diabetes, a β cell compensatoryhypersecretion of insulin occurs and this process is accompanied by cellsurface expression of the molecule Fas. When NOD mice, an animal modelfor Type I diabetes, are crossed with mice having the lpr mutation (Fasdeficient), the animals are resistant to disease. In addition,destruction of β cells in the NOD accelerates when Fas Ligand is placedon the insulin promotor.

It has been discovered according to the invention that changes inmitochondrial metabolic processes that alter mitochondrial membranepotential and Fas expression contribute to Fas-induced β celldestruction. β cell glucose-induced insulin secretion depends onincreased intracellular ATP. The mitochondrial synthesis of ATP occursthrough coupling of electron transport-dependent oxido-reductivereactions to ATP synthetase (oxidative phosphorylation). During thisprocess, a proton gradient is generated by the pumping of protons out ofthe mitochondria increasing mitochondrial membrane potential. Uncouplingproteins (UCPs) reversibly uncouple oxidative phosphorylation fromelectron transport decreasing mitochondrial membrane potential. Normalpancreatic β cell are in an uncoupled state and do not express Fas ontheir cell surface. As diabetes progresses to a first stage in which thepatient is sick but before the pancreatic β cell are destroyed, thepatients pancreatic β cells become coupled and express Fas on the cellsurface. The disease then progresses to the stage when pancreatic β cellbegin to be killed. Before the cells are killed the metabolic statechanges again to uncoupled and Fas is still expressed on the surface.When the cell is in an uncoupled state and Fas is expressed on the cellsurface the cell is killed as soon as Fas is engaged without the needfor any other agents.

The methods of the invention include a method for inhibiting pancreaticβ cell death in a Type I diabetic by altering the mitochondrialmetabolic state. The method is performed by contacting a pancreatic βcell of a Type I diabetic with an amount of a metabolic modifying agenteffective to increase mitochondrial membrane potential in the pancreaticβ cell. The metabolic modifying agent causes the pancreatic β cell torevert to or remain in a coupled state. Although these cells are not inthe normal state of a pancreatic β cell, they are not killed and thepatients organ is not destroyed.

Another method for inhibiting the death of a pancreatic β cell in a TypeI diabetic can be accomplished by contacting a pancreatic β cell of aType I diabetic with an amount of a Fas binding agent effective toinhibit selective engagement of Fas on the surface of the pancreatic βcell. By inhibiting the selective engagement of Fas on the cell surfaceand allowing the cell to remain in the uncoupled state the cell willremain healthy and have the phenotype of a normal pancreatic β cell.

The Fas binding agents which are useful according to the invention arethose molecules which bind to Fas but do not activate it. Fas bindingagents can be identified by screening libraries using the extracellularregions of Fas, such as the screening methods described below. Fasbinding agents then can easily be tested without undue experimentationin vitro for their ability to bind Fas but not induce cell death inuncoupled cells. Uncoupled cells can be prepared according to themethods described above. Fas can be induced to be expressed on thesurface of cells using irradiation as has previously been identified inthe prior art. Once uncoupled cells expressing Fas have been developedpotential Fas binding agents can be incubated with these cells and celllysis can be assayed by the methods described herein or by other methodsknown in the art.

The invention is also useful for treating type II diabetics. A “type IIdiabetic” is a subject who has diabetes mellitus caused by abnormalinsulin secretion and/or resistance to insulin action in target tissues.The physiological problem which occurs in a Type II diabetic is verydifferent than that which occurs in a type I diabetic. In type IIdiabetes the pancreatic β cells undergo excessive proliferation. It isdesirable to inhibit proliferation of these cells.

One method for inducing pancreatic β cell death in a Type II diabeticinvolves the step of contacting a pancreatic β cell of a Type IIdiabetic with an amount of an MHC class II HLA-DR inducing agenteffective to induce the expression of the MHC class II HLA-DR on thesurface of the pancreatic β cell, and selectively engaging the MHC classII HLA-DR by contacting the cell with an MHC class II HLA-DR ligandeffective to induce pancreatic β cell death.

Another finding according to the invention was that mitochondrialmetabolism and the related expression of MHC class II on the surface ofa cell is indicative of the susceptibility of the host of that cell todeveloping atherosclerosis, autoimmune disease or multiple sclerosis.When electron transport and oxidative phosphorylation are in a coupledstate in a cell the cell expresses MHC class II HLA-DP/DQ on thesurface. When electron transport and oxidative phosphorylation are in anuncoupled state in a cell the cell expresses MHC class II HLA-DR on thesurface. A cell in a coupled state that has MHC class II HLA-DP/DQ onthe surface will be stimulated to divide when the MHC class II HLA-DP/DQis engaged. A cell in an uncoupled state that has MHC class II HLA-DR onthe surface will be stimulated to lyse when the MHC class II HLA-DR isengaged.

These different metabolic states of the cell have been found accordingto the invention to be predictive of an individuals susceptibility todeveloping disease. When the cells of a subject are coupled and expressMHC class II HLA-DP/DQ on the surface the subject is susceptible todeveloping atherosclerosis. When the cells of a subject are uncoupledand express MHC class II HLA-DR on the surface the subject issusceptible to developing autoimmune disease.

The invention encompasses methods for screening a subject forsusceptibility to atherosclerosis. These methods involve the steps ofisolating a cell which is useful for screening such as a peripheralblood lymphocyte or a skin cell from a subject and detecting thepresence of an MHC marker selected from the group consisting of an MHCclass II HLA-DP/DQ, B7-2, B7-1 and MHC class II HLA-DR on the surface ofperipheral blood lymphocyte, wherein the presence of MHC class IIHLA-DP/DQ is indicative of susceptibility to atherosclerosis and thepresence of MHC class II HLA-DR is indicative of resistance toatherosclerosis.

Atherosclerosis is a group of diseases affecting the cardiovascularsystem and includes myocardial infarction, stroke, angina pectoris andperipheral cardiovascular disease. Despite significant advices intherapy, cardiovascular disease remains the single most common cause ofmorbidity and mortality in the developed world. Many individuals aresusceptible to developing future cardiovascular disorders, and thissusceptibility has usually been defined in terms of risk factors such asfamily history of premature ischemic heart disease, hyperlipidemia,cigarette smoking, hypertension, low HDL cholesterol, diabetes mellitus,hyperinsulinemia, abdominal obesity, and high lipoprotein. The inventionincludes a new method for determining an individuals susceptibility todeveloping atherosclerosis. As used herein susceptibility toatherosclerosis indicates a likelihood of 10% greater than the averageof developing atherosclerosis.

The invention also encompasses methods for screening a subject forsusceptibility to autoimmune disease. These methods involve the steps ofisolating a peripheral blood lymphocyte from a subject and detecting thepresence of an MHC marker selected from the group consisting of an MHCclass II HLA-DP/DQ, B7-2, B7-1 and MHC class II HLA-DR on the surface ofperipheral blood lymphocyte, wherein the presence of MHC class II HLA-DRis indicative of susceptibility to autoimmune disease and the presenceof MHC class II HLA-DP/DQ is indicative of resistance to autoimmunedisease.

Autoimmune disease is a class of diseases in which an individuals ownantibodies react with host tissue or in which immune effector T cellsare autoreactive to endogenous self peptides and cause destruction oftissue. It is well established that MHC class II alleles act as majorgenetic elements in susceptibility to a variety of autoimmune diseases.These include rheumatoid arthritis, celiac disease, pemphigus vulgaris,and the prototype for autoimmune disease, systemic lupus erythematosus(SLE). The invention includes a new method for determining anindividuals susceptibility to developing autoimmune disease. As usedherein susceptibility to Autoimmune disease indicates a likelihood of10% greater than the average of developing autoimmune disease.

The methods of the invention also include methods for treating a subjecthaving autoimmune disease to reduce associated cell death. One method isbased on the interaction between cells expressing MHC class II HLA-DRand γδ T cells. γδ T cells specifically recognize MHC class II HLA-DR onthe surface of the cell and stimulate cell death. When the γδ T cellsrecognize a tissue having significant amounts of MHC class II HLA-DRthese T cells become activated and proliferate in order to kill more ofthe recognized cells. The methods of treatment are based on the conceptof eliminating the activated γδ T cells from the body. These cells canbe removed by isolating a sample of peripheral blood and identifying theactivated γδ T cells by assessing activation markers using flowcytometry. Antibodies can then be generated to the specific activated γδT cells and the antibodies can be used to selectively bind to andinactivate γδ cells in the subject. This inactivation of the γδ cellsinhibits cell death associated with autoimmune disease.

Similarly cells expressing cell surface MHC class II HLA-DR that areordinarily recognized and killed by γδ T cells can be used for thetreatment of diseases involving excessive cell proliferation such asglioma. The cells can be induced to undergo cell death by stimulatingexcess activated γδ T cells in the subject. This can be accomplishedusing bacterial byproducts.

It has been found according to the invention that a link exists betweenFas expression, mitochondrial metabolism, and susceptibility toFas-dependent cell death. Thus by regulating mitochondrial metabolism itis possible to control susceptibility to Fas dependent cell death. Thisphenomenon is described below with respect to pancreatic β cells, but isapplicable to all biological systems described herein.

Type I diabetes mellitus (DM) is a pancreatic β cell-selectiveautoimmune disease which results in insulin deficiency. Neither thegenetic/environmental influences nor the inherent β cell characteristicsthat trigger immune-mediated destruction are completely understood.Apoptosis has been suggested as the mechanism of β cell death in mousemodels of Type I diabetes. Two features that correlate withsusceptibility to β cell destruction are the metabolic state of the βcells and expression of the cell surface molecule Fas (CD95), a memberof the TNF family of “death inducing” receptor/ligand pairs. During theprediabetic stage of Type I DM, a β cell glucose-dependent hypersectionof insulin occurs in response to high glucose concentrations and thisprocess is coincident with the cell surface expression of Fas. When NODmice are crossed with mice having the lpr mutation (Fas deficient), theanimals are resistant to disease. In addition, destruction of β cells inthe NOD accelerates when Fas ligand is placed on the insulin promoter.In the NOD model, apoptotic β cells have been observed in the islets at15 weeks of age which coincides with the earliest onset of diabetes asdetermined by blood glucose, urine glucose, and pancreaticimmunoreactive insulin measurements. The incidence of apoptosisdecreases by week 18 at which time 50% of the animals have overtdiabetes. Virtually all of the apoptotic cells have been determinedimmunohistochemically to be positive for insulin production.Interestingly, apoptosis of β cells precedes the appearance of T cellsin islets. The ability to upregulate Fas expression on β cells is alsoacquired during the early stages of Type I DM.

It is believed according to the invention that the metabolic state ofthe β cell determines the susceptibility of β cells to Fas mediateddeath. β cell glucose-induced insulin secretion depends upon increasedintracellular ATP. The mitochondrial synthesis of ATP results from thecoupling of electron transport-dependent oxido-reductive reactions toATP synthetase (oxidative phosphorylation). During this process, aproton gradient is generated by the pumping of protons across themitochondrial membrane resulting in an increase in mitochondrialmembrane potential. Uncoupling proteins (UCP) can reversibly dissipatethe proton gradient resulting in decreased membrane potential.Mitochondrial damage, resulting from viruses, inflammation, age, oroxidative stress, can also dissipate the proton gradient and decreasethe mitochondrial membrane potential. However, in the latter case, thechange in mitochondrial metabolism is irreversible. For example,increased intracellular NO production in β cells is known to alter βcell mitochondrial membrane potential and sensitize β cells toFas-induced death. Our data (provided in the Examples below) demonstratethat β cells express intracellular UCP. Furthermore, we have shown thatβ cell surface Fas expression and mitochondrial membrane potentialincrease as a function of environmental glucose concentration. Takentogether, these results are consistent with the notion thatmitochondrial glucose metabolism and consequent mitochondrial membranepotential play a critical regulatory role in susceptibility toFas-induced β cell death.

Increasing environmental glucose results in increased cell surface Fasexpression and functionally coupled mitochondrial ATP synthesis,suggesting a link between mitochondrial glucose metabolism andsusceptibility to Fas-induced cell death. ATP is required for insulinsecretion. As glucose levels decrease, levels of cell surface Fasdecrease, newly synthesized Fas is stored intracellularly andmitochondrial ATP synthesis is uncoupled from respiration and lessmitochondrial ATP is produced. This is demonstrated schematically inFIG. 14. The reversibility of this process may account for thepulsatility of insulin secretion in response to nutrients. In eitherstate, coupled or uncoupled, damaging agents such as diabetogenicviruses, inflammation, ischemia, age, or oxidative stress, may damagemitochondrial metabolism, increase cell surface Fas expression, andrender the cells susceptible to Fas-induced apoptosis or oncosis,respectively. One possibility is that during the insulitis phase of TypeI DM, apoptosis (on the right of the panel), which is thought to occur“silently” without additional inflammation, occurs to some of the βcells and that oncosis occurs in later stages of disease resulting fromT cell mediated (FasL dependent) β cell destruction.

The invention in other aspects relates to methods for selectivelykilling a Fas ligand bearing tumor cell. The method involves the stepsof contacting the Fas ligand bearing tumor cell with acetate in anamount effective to induce Fas associated cell death. A Fas ligandbearing tumor cell is any tumor cell which inducibly or constitutivelyexpressed a Fas ligand on the cell surface. Such cells can easily beidentified by those of skill in the art since the Fas ligand is a wellknown molecule. These cells include but are not limited to melanomacells and colon carcinoma cells.

Although acetate alone is sufficient to kill a Fas ligand bearing tumorcell, the cell can also be treated with a chemotherapeutic agent and/ora Fas ligand to promotes killing. The use of these secondary compoundsallows the use of less of the acetate to be used to accomplish the cellkilling. The combination of acetate and chemotherapeutic agents and orFas ligands, allows less of all three reagents to be used than wouldotherwise be required to kill the cell.

Additionally, tumor cells that do not express cell surface Fas ligandcan also be killed by the methods of the invention. This killing can beaccomplished by contacting the tumor cell with acetate in an amounteffective to induce cell surface Fas expression, and administering a Fasligand to the tumor cell in an amount effective to induce Fas associatedcell death. Fas ligands are expressed on the surface of NK γδ T cells,CD4 T cells, CD8 T cells, etc.

Other methods for selectively killing a cell include contacting the cellwith a nucleic acid selected form the group consisting of a UCPanti-sense nucleic acid and a UCP dominant-negative nucleic acid in anamount effect to inhibit UCP function. A cell can also be killedaccording to the invention by contacting the cell with a compoundselected from the group consisting of acetate and GDP and an apoptopicchemotherapeutic agent in an amount effective to kill the cell.

The invention also encompasses methods for promoting a Th1 immuneresponse. The method is performed by administering to a subject who hasbeen exposed to an antigen an effective amount for inducing a Th1 immuneresponse of a MHC class II HLA-DR inducing agent to induce DR on a Tcell. MHC class II HLA-DR inducing agents are discussed in detail above,and include, for instance, fatty acids.

The invention in another aspect is a method for inducing nerve celldifferentiation by contacting a nerve cell with an amount of a B7inducing agent effective to induce the expression of B7 on the surfaceof the nerve cell and exposing the nerve cell to a neural activatingcell to cause differentiation of the nerve cell.

The complex process of immune cell activation and proliferation is basedon diverse interactions such as antigen presentation, cell-cell contactand soluble immune mediators e.g., cytokines or lymphokines. Many ofthese interactions are mediated in T—and other immune cells throughsurface receptors. T helper cells, for example, require for activationboth the presentation of an antigen by an antigen presenting cell (APC)in association with major histocompatibility complex (MHC) and asecondary signal. The secondary signal may be a soluble factor or mayinvolve an interaction with another set of receptors on the surface ofT—and other immune cells. Antigen presentation in the absence of thesecondary signal, however, is not sufficient to activate T helper cells.

The CTLA-4/CD28/B7 system is a group of proteins involved in regulatingT-cell proliferation through this secondary signaling pathway. TheT-cell proliferative response is controlled by the interaction of the B7family of proteins, which are expressed on the surface of APCs, withCTLA-4 (cytotoxic T lymphocyte antigen #4) and CD28.

The B7 family of proteins is composed of structurally relatedglycoproteins including B7-1, B7-2, and B7-3 (Galea-Lauri et al., CancerGene Therapy, v. 3, p. 202-213 (1996); Boussiotis, et al., Proc. Nat.Acad. Sci. USA, v. 90, p. 11059-11063 (1993)). The different B7 proteinsappear to have different expression patterns on the surface of antigenpresenting cells. For example B7-2 is constitutively expressed on thesurface of monocytes, whereas B7-1 is not, although B7-1 expression isinduced in these cells when the cells are stimulated with interferongamma (IFN-γ). The different expression patterns may indicate adifferent role for each of the B7 family members. The B7 proteins arebelieved to be involved in the events relating to stimulation of animmune response by its ability to interact with various immune cellsurface receptors. It is believed, for example, that B7 plays a role inaugmenting T-cell proliferation and cytokine production through itsinteraction with the CD28 receptor.

CD28, a homodimeric glycoprotein having two disulfide linked 44-kdsubunits, is found on 95% of CD4⁺ and 50% of CD8⁺ cells. Studies usingmonoclonal antibodies reactive with CD28 have demonstrated that CD28 isinvolved in a secondary signal pathway in the activation of T-cellproliferation. Antibodies which block the interaction of CD28 with itsligand have been found to inhibit T-cell proliferation in vitroresulting in antigen specific T cell anergy. (Harding et al., Nature, v.356, p. 607 (1991)).

Recently a T-cell surface receptor protein, CTLA-4, having approximately20% sequence homology to CD28 was identified. Although CTLA-4 is notendogenously expressed on T-cell surfaces, its expression is inducedwhen CD28 interacts with B7 on the surface of an APC. Once CTLA-4 isexpressed on the surface of the T-cell it is capable of interacting withB7.

It was discovered according to one aspect of the invention that nervecells can be induced to express B7 and can interact with T—and otherimmune cells through B7/CD28/CTLA4 molecules. The B7 on the nerve cellsurface can engage the CD28/CTLA4 on the immune cell surface toco-stimulate the immune cell, leading to activation of the immune cell.The activated immune cell then releases nerve growth factor whichstimulates the nerve cell.

As used herein “B7 inducing agent” is an agent which causes B7 (andother related family members retaining sequence homology with B7) to beexpressed on a nerve cell surface. In one preferred embodiment the B7inducing agent is a pharmacological agent that causes dissipation ofproton motor force such as by uncoupling of electron transport andoxidative phosphorylation, resulting in reduced mitochondrial membranepotential within the cell. B7 inducing agents which cause dissipation ofthe proton motor force include but are not limited to adriamycin, gammainterferon, bacterial byproducts such as lipopolysaccharides,lipoproteins BCG, fatty acids, cAMP inducing agents and a UCP expressionvector. A “cAMP inducing agent” as used herein is any compound whichelevates intracellular levels of cAMP. Such compounds include but arenot limited to isoproterenol, epinephrine, norepinephrine,phosphodiester inhibitors, theophylline, and caffeine. In anotherpreferred embodiment the B7 inducing agent is a B7 expression vector.Such a vector can be stably expressed in the nerve cell to produce B7which can be expressed on the cell surface. The B7 inducing agent is anisolated molecule. An isolated molecule is one which has been removedfrom its natural surroundings and formulated for administration to anorganism.

An “amount of a B7 inducing agent effective to induce the expression ofB7 on the surface of the nerve cell” as used herein, refers to an amountwhich is effective to cause dissipation of a proton motor force and thusto decrease the mitochondrial membrane potential in the nerve cell.Preferably the amount is that amount which is necessary to induce theexpression of at least a single B7 molecule on the cell surface.

The nerve cell is contacted with the B7 inducing agent to causeexpression of B7 on the surface. As used herein, the step of contactingthe cell with B7 inducing agent can be performed by any means known inthe art. For instance, if the B7 inducing agent is applied in vitro, itmay simply be added as part of the cellular medium to a tissue culturedish of nerve cells. If the method is performed in vivo, then the stepof contacting may be performed by administering the B7 inducing agent bycommonly used therapeutic techniques, such as parenteral administration,oral administration, or local administration. Other methods are wellknown to those of ordinary skill in the art.

According to a method of the invention the B7 expressing nerve cell isexposed to a neural activating cell. A “neural activating cell” as usedherein, is a cell which is capable of producing nerve growth factor whenactivated and which includes a cell surface B7 receptor. As mentionedabove, B7 receptors include CD28 and CTLA-4. Many cells which are theneural activating cells of the invention have been described in theprior art. These cells include, for example, T cells (including bothgamma, delta and alpha-beta T cells), macrophage, dendritic cells,CTLA-4 or CD-28 expressing B cells.

A “B7 receptor” as used herein is a cell surface immune molecule whichinteracts with B7 on a partner cell and cases activation of the cell onwhich it is expressed. Preferably the B7 receptor is a CD28 molecule ora CTLA4 molecule.

The nerve cell is exposed to the neural activating cell to causedifferentiation of the nerve cell. The step of exposing can be performedin vitro, by simply mixing the two populations of cells, the nerve celland the neural activating cell. It can be accomplished in vivo bycausing the accumulation of the neural activating cells in the localenvironment of the nerve cell. For instance, the neural activating cellsmay be implanted, or the local environment may be manipulated to causeaccumulation of the neural activating cell. For instance, stimulating animmune response in the local environment would cause the accumulation ofT cells, B cells, dendritic cells and macrophage. The neural activatingcell may also be a cell which produces nerve growth factor uponactivation and which is engineered to express a B7 receptor on itssurface, e.g. by transfection with an inducible or constitutivelyexpressed B7 receptor gene, such as by the methods described above.

The methods of the invention in some aspects may also be performed usingan endogenous neural activating cell. For instance the endogenous neuralactivating cell may be a cell having a cell surface B7 receptor, such asCD28 and CTLA-4. In this case the method would only include the step ofcontacting a nerve cell with an amount of a B7 inducing agent effectiveto induce the expression of B7 on the surface of the nerve cell in thepresence of a neural activating cell.

When the neural activating cell is a cell having a cell surface B7receptor which is already present in interactive proximity to the B7,the cell does not have to be manually brought into contact with the B7on the nerve cell.

When the nerve cell is exposed to a neural activating cell the cellsurface B7 can interact with the B7 receptor to activate the neuralactivating cell. Once activated, the neural activating cell produces andreleases nerve growth factor into the local environment. This locallyproduced nerve growth factor is capable of causing the nerve cell tobecome differentiated. Although the invention is not limited to aspecific mechanism of action, applicants believe that the mechanismthrough which neuro-differentiation occurs is that the nerve growthfactor interacts with the nerve cell surface nerve growth factorreceptor such as Trk. It is also believed that engagement of the B7 onthe cell surface or the induction thereof causes the expression of nervegrowth factor receptors on the surface of the nerve.

In one embodiment of the invention, the receptors for nerve growthfactor may be induced to be expressed on the surface of the nerve cell.Two known nerve growth factors are tyrosine, kinase A (TrkA) andp75NGRF. When these receptors interact with nerve growth factor on thesurface of a nerve cell, it stimulates the cell to undergo neuronaldifferentiation. Expression of these receptors on the surface of thenerve cell may be performed by any method known in the art. Forinstance, the nerve cell may be recombinantly engineered toconstitutively or inducibly express the DNA for these receptors, such asby the methods described above.

Nerve growth factor (NGF), originally described by Levi-Montalcini andHamburger in 1953 (Levi-Montalcini and Hamburger, 1953), contains twocopies of three types of polypeptides designated α, β and γ and exhibitsapproximately 50% of homology with other neurotrophins i.e.,brain-derived neurotrophic factor (BDNF), NT-3, NT-4 and NT-5 (Siegel etal., 1994). It binds to tyrosine kinase A (TrkA) and p75NGF receptors ina synergistic manner (Canossa et al., 1996). Tyrosine kinase B (TrkB)and tyrosine kinase C (TrkC) receptors preferentially bind BDNf and NT-3respectively (Siegel et al., 1994). Intracellular signal proteins viaSrc homology 2 (SH20 domain interactions such as phospholipase C-γ andthe p85 sub-unit of phosphatidyl-inositol 3-kinase bind to thetyrosine-phosphorylated receptors and allow multimeric protein complexesto form and lead to the activation of specific signal transductionpathways (Hempstead et al., 1994).

As shown in the Examples below, nerve cells express molecules which arerequisite for T cell activation, indicating that there is aneuro-immunological intercellular interactive component that occursduring neuronal differentiation. NGF and EGF have profound effects onthe differentiation process in utero and early life and on theregeneration process after pathologic damage. The data provided in theexamples is relevant since it not only demonstrates the existence ofinducible surface molecules on post-mitotic neurons, but their abilityto be kinetically modified by the presence or absence of specifictrophic factors is also highlighted. The presence of Fas on the neuronalcell surface suggests that PC12 cells and their variants are vulnerableto apoptosis or that the molecule is capable of transmitting a mitoticsignal if required.

Another aspect of the invention involves a method for inducing apoptosisin a nerve cell. The method involves the step of contacting a nerve cellwith an amount of a metabolic modifying agent which when exposed to anerve cell causes coupling of electron transport and oxidativephosphorylation effective to increase the mitochondrial membranepotential in the nerve cell and contacting a neural activating cell withan amount of a B7 receptor blocking agent effective for inducingapoptosis in the nerve cell. “Metabolic modifying agents” and “Fasbinding agents” are discussed above.

A “B7 receptor blocking agent” as used herein is any agent whichinteracts with a B7 receptor but does not cause activation of the celland prevents that receptor from binding to B7. These agents include, forexample, but are not limited to anti-CD28 antibodies, CD28 bindingpeptides, anti-CTLA-4 antibodies, CTLA-4 analogs and CTLA-4 bindingpeptides which do not cause activation of the receptor. Other B7receptor blocking agents can be identified by those of skill in the artby routine experimentation using immune cell activation assays such as aT cell activation assay.

This method is useful whenever it is desirable to induce apoptosis of anerve cell. For instance, it may be useful to induce apoptosis of anerve cell in vitro in order to screen molecules for their ability toprevent apoptosis of nerve cells. Other uses will be apparent to thoseof ordinary skill in the art.

As discussed above, when a cell is coupled, Fas is expressed on the cellsurface and when a cell is uncoupled Fas generally is transported tointracellular stores. When a cell is coupled and Fas is on the surfaceengagement of Fas sends a signal to the cell instructing the cell toundergo cellular division. When a cell is uncoupled ordinarily Fas isnot expressed on the cell surface. In the presence of NGF, however, Fasis down regulated and is no longer expressed on the cell surface. In adamaged tissue if a nerve cell is in an uncoupled state, and expressesboth Fas and B7 on the surface, then the presence or absence of NGF willdetermine the fate of the cell. If an NGF producing cell according tothe invention is present in the local environment, the B7 of the nervecell will stimulate production of NGF by interacting with that cell. Thelocal NGF produced will cause the down regulation of Fas and the cellwill undergo differentiation. If an NGF producing cell is not availableor if B7 is not expressed on the surface of the nerve cell, thenenvironmental factors can stimulate Fas to cause apoptosis.

Another aspect of the invention is a method for reinnervating an injuredtissue. The method involves the step of implanting a B7 expressing nervecell in the injured tissue, wherein the implanted B7 expressing nervecell will undergo neuronal differentiation in the presence of a neuralactivating cell in the injured tissue to reinnervate the injured tissue.Methods are known in the art implanting nerve cells into living tissue.For example, nerves can be implanted directly into exposed tissue or maybe implanted in biodegradable tubes which will guide the extension ofthe nerve into surrounding tissue where it can be differentiated.

A B7 expressing nerve cell can be prepared by an means known in the art.For instance, a B7 expressing nerve cell may be genetically engineeredto constitutively or inducibly express B7. The gene encoding a B7protein can be constitutively expressed in a nerve cell by transfectionprocedures known in the art, such as by the methods described above. B71gene is provided herein as SEQ ID No. 1 and listed under Accession No.M27533 in Genebank and the nucleic acid sequence for B72 is providedherein as SEQ ID No. 2 and listed under Accession No. U04343 inGenebank. Alternatively the nerve cell may be engineered to inducibly orconstitutively express UCP which will induce expression of endogenousB7. In another embodiment, the implanted B7 expressing nerve cell mayalso constitutively or inducibly express at least one of the nervegrowth factor receptors, which would induce expression of endogenous B7.

An injured tissue is a tissue in which nerve damage has been sustained.An injured tissue may include for example, a spinal chord injury, asevered or severely damaged limb or any other tissue which can beinnervated and in which the nerve has been damaged. Neural activatingcells are generally found in skin and muscle surrounding the nerves ofan injured tissue. These neural activating cells can stimulate thedifferentiation of the nerve cell once they are activated by interactionwith the B7 on the surface of the nerve cell.

The invention also includes a method for treating a neurodegenerativedisorder by administering an amount of a B7 inducing agent effective toinduce the expression of B7 on the surface of a nerve cell. An amountthat is effective to induce the expression on the surface is an amountwhich is effective to cause dissipation of a proton motor force and thusto decrease the mitochondrial membrane potential in the nerve cell.

A “neurodegenerative disorder” as used herein, is a disorder associatedwith the death or injury of neuronal cells. For example, the loss ofdopaminergic neurons in the substantia nigra ultimately leads toParkinson's Disease. The deposition of β-amyloid protein in the braingenerally causes neural damage leading to Alzheimer's Disease.Conditions involving injuries such as brain ischemia, spinal chorddamage, and severance of limbs often causes extensive neuronal celldeath. When a nerve is severed, the regions of the nerve cells which aredistal to the severance become separated from the nerve cell body anddegenerate. After such a severance, it is possible for the nerve cellbody to regenerate by re-extension of the severed axons. This process ofnerve regeneration does not occur naturally in the absence of certainenvironmental conditions. In some cases in the prior art, variousfactors such as nerve growth factor have been added to the nerve toattempt to stimulate the regeneration. The methods of the inventiondescribe a different system in which the nerve cell is manipulated toexpress an immune recognition molecule on its surface which can thencause the local expression of nerve growth factor leading todifferentiation. This method more closely simulates the naturalprocesses of neuronal regeneration. Other neurodegenerative diseasesinclude for example but are not limited to epileptic seizures andamyotrophic lateral sclerosis.

The invention also includes compositions of the above described agents.One composition of the invention includes a metabolic modifying agentand an apoptotic chemotherapeutic agent. The pharmaceutical preparationsof the invention are administered to subjects in effective amounts. Aneffective amount means that amount necessary to delay the onset of,inhibit the progression of, halt altogether the onset or progression ofor diagnose the particular condition being treated. In one embodimentthe metabolic modifying agent and the apoptotic chemotherapeutic agentare present in an effective dose for treating a tumor. In anotherembodiment the metabolic modifying agent and the apoptoticchemotherapeutic agent are present in an effective dose for treatingtype II diabetes. In general, an effective amount for treating cancerand type I diabetes will be that amount necessary to favorably affectmammalian cell proliferation in-situ. When administered to a subject,effective amounts will depend, of course, on the particular conditionbeing treated; the severity of the condition; individual patientparameters including age, physical condition, size and weight;concurrent treatment; frequency of treatment; and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

Another composition according to the invention is an MHC class II HLA-DRinducing agent and an MHC class II HLA-DR ligand. In one embodiment theMHC class II HLA-DR inducing agent and MHC class II HLA-DR ligand arepresent in an effective dose for treating type II diabetes. In general,an effective amount for treating type II diabetes will be that amountnecessary to favorably affect mammalian cell proliferation in-situ. Whenadministered to a subject, effective amounts will depend, of course, onthe particular condition being treated; the severity of the condition;individual patient parameters including age, physical condition, sizeand weight; concurrent treatment; frequency of treatment; and the modeof administration. These factors are well known to those of ordinaryskill in the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

One composition of the invention is a B7 inducing agent and a B7receptor inducing agent. In one embodiment the B7 inducing agent and B7receptor inducing agent present in an effective dose for treatingneurodegenerative disease. In general, an effective amount forneurodegenerative disease will be that amount necessary to favorablyaffect nerve cell differentiation in-situ. When administered to asubject, effective amounts will depend, of course, on the particularcondition being treated; the severity of the condition; individualpatient parameters including age, physical condition, size and weight;concurrent treatment; frequency of treatment; and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

Generally, doses of active compounds will be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses range of 50-500mg/kg will be suitable, in one or several administrations per day. Inthe event that a response in a subject is insufficient at the initialdoses applied, higher doses (or effectively higher doses by a different,more localized delivery route) may be employed to the extent thatpatient tolerance permits. Multiple doses per day are contemplated toachieve appropriate levels of compounds.

The invention involves the use of several different types of bindingpeptides or molecules, MHC class II HLA-DR binding peptides, CD4/αβTCRbinding molecules, CD40 binding peptides, MHC class II HLA-DP/DQ bindingpeptides, CD28/CTLA4 binding peptides, and Fas biding peptides. Thebinding peptides of the invention can be identified using routineassays, such as the binding and activation assays described in theExamples and elsewhere throughout this patent application.

The binding peptides of the invention are isolated peptides. As usedherein, with respect to peptides, the term “isolated peptides” meansthat the peptides are substantially pure and are essentially free ofother substances with which they may be found in nature or in vivosystems to an extent practical and appropriate for their intended use.In particular, the peptides are sufficiently pure and are sufficientlyfree from other biological constituents of their hosts cells so as to beuseful in, for example, producing pharmaceutical preparations orsequencing. Because an isolated peptide of the invention may be admixedwith a pharmaceutically acceptable carrier in a pharmaceuticalpreparation, the peptide may comprise only a small percentage by weightof the preparation. The peptide is nonetheless substantially pure inthat it has been substantially separated from the substances with whichit may be associated in living systems.

The binding peptides also may easily be synthesized or produced byrecombinant means by those of skill in the art. Methods for preparing oridentifying peptides which bind to a particular target are well known inthe art. Molecular imprinting, for instance, may be used for the de novoconstruction of macromolecular structures such as peptides which bind toa particular molecule. See for example Kenneth J. Shea, MolecularImprinting of Synthetic Network Polymers: The De Novo synthesis ofMacromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem. Sci.,19(9) January 1994; and Wulff, G., in Polymeric Reagents and Catalysts(Ford, W. T., Ed.) ACS Symposium Series No. 308, pp 186-230, AmericanChemical Society (1986). One method for preparing mimics of the knownbinding peptides involves the steps of: (i) polymerization of functionalmonomers around a known binding peptide or the binding region of anantibody which also binds to the targets (the template) that exhibits adesired activity; (ii) removal of the template molecule; and then (iii)polymerization of a second class of monomers in the void left by thetemplate, to provide a new molecule which exhibits one or more desiredproperties which are similar to that of the template. In addition topreparing peptides in this manner other binding molecules which have thesame function as the binding peptides useful according to the inventionsuch as polysaccharides, nucleosides, drugs, nucleoproteins,lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and otherbiologically active materials can also be prepared. This method isuseful for designing a wide variety of biological mimics that are morestable than their natural counterparts, because they are typicallyprepared by the free radical polymerization of functional monomers,resulting in a compound with a nonbiodegradable backbone. Other methodsfor designing such molecules include for example drug design based onstructure activity relationships which require the synthesis andevaluation of a number of compounds and molecular modeling.

The binding peptides may also be identified by conventional screeningmethods such as phage display procedures (e.g., methods described inHart, et al., J. Biol. Chem. 269:12468 (1994)). Hart et al. report afilamentous phage display library for identifying novel peptide ligandsfor mammalian cell receptors. In general, phage display libraries using,e.g., M13 or fd phage, are prepared using conventional procedures suchas those described in the foregoing reference. The libraries displayinserts containing from 4 to 80 amino acid residues. The insertsoptionally represent a completely degenerate or a biased array ofpeptides. Ligands having the appropriate binding properties are obtainedby selecting those phages which express on their surface a ligand thatbinds to the target molecule. These phages then are subjected to severalcycles of reselection to identify the peptide ligand-expressing phagesthat have the most useful binding characteristics. Typically, phagesthat exhibit the best binding characteristics (e.g., highest affinity)are further characterized by nucleic acid analysis to identify theparticular amino acid sequences of the peptides expressed on the phagesurface and the optimum length of the expressed peptide to achieveoptimum binding. Alternatively, such peptide ligands can be selectedfrom combinatorial libraries of peptides containing one or more aminoacids. Such libraries can further be synthesized which containnon-peptide synthetic moieties which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts.

To determine whether a peptide binds to the appropriate target any knownbinding assay may be employed. For example, in the case of a peptidethat binds to the MHC class II LA-DR the peptide may be immobilized on asurface and then contacted with a labeled MHC class II HLA-DR (or viceversa). The amount of MHC class II HLA-DR which interacts with thepeptide or the amount which does not bind to the peptide may then bequantitated to determine whether the peptide binds to MHC class IIHLA-DR. A surface having a known peptide that binds to MHC class IILA-DR such as a commercially available monoclonal antibody immobilizedthereto may serve as a positive control.

Screening of peptides of the invention, also can be carried oututilizing a competition assay. If the peptide being tested competes withthe known monoclonal antibody, as shown by a decrease in binding of theknown monoclonal antibody, then it is likely that the peptide and theknown monoclonal antibody bind to the same, or a closely related,epitope. Still another way to determine whether a peptide has thespecificity of the known monoclonal antibody is to pre-incubate theknown monoclonal antibody with the target with which it is normallyreactive, and then add the peptide being tested to determine if thepeptide being tested is inhibited in its ability to bind the target. Ifthe peptide being tested is inhibited then, in all likelihood, it hasthe same, or a functionally equivalent, epitope and specificity as theknown monoclonal antibody.

By using the known MHC class II HLA-DR (and other target) monoclonalantibodies of the invention, it is also possible to produceanti-idiotypic antibodies which can be used to screen other antibodiesto identify whether the antibody has the same binding specificity as theknown monoclonal antibody. Such anti-idiotypic antibodies can beproduced using well-known hybridoma techniques (Kohler and Milstein,Nature, 256:495, 1975). An anti-idiotypic antibody is an antibody whichrecognizes unique determinants present on the known monoclonalantibodies. These determinants are located in the hypervariable regionof the antibody. It is this region which binds to a given epitope and,thus, is responsible for the specificity of the antibody. Ananti-idiotypic antibody can be prepared by immunizing an animal with theknown monoclonal antibodies. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing known monoclonalantibodies and produce an antibody to these idiotypic determinants. Byusing the anti-idiotypic antibodies of the immunized animal, which arespecific for the known monoclonal antibodies of the invention, it ispossible to identify other clones with the same idiotype as the knownmonoclonal antibody used for immunization. Idiotypic identity betweenmonoclonal antibodies of two cell lines demonstrates that the twomonoclonal antibodies are the same with respect to their recognition ofthe same epitopic determinant. Thus, by using anti-idiotypic antibodies,it is possible to identify other hybridomas expressing monoclonalantibodies having the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is theimage of the epitope bound by the first monoclonal antibody.

In one embodiment the binding peptides useful according to the inventionare antibodies or functionally active antibody fragments. Antibodies arewell known to those of ordinary skill in the science of immunology. Manyof the binding peptides described herein are available from commercialsources as intact functional antibodies. As used herein, the term“antibody” means not only intact antibody molecules but also fragmentsof antibody molecules retaining specific binding ability. Such fragmentsare also well known in the art and are regularly employed both in vitroand in vivo. In particular, as used herein, the term “antibody” meansnot only intact immunoglobulin molecules but also the well-known activefragments F(ab′)₂, and Fab. F(ab′)₂, and Fab fragments which lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding of an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

As is well-known in the art, the complementarity determining regions(CDRs) of an antibody are the portions of the antibody which are largelyresponsible for antibody specificity. The CDR's directly interact withthe epitope of the antigen (see, in general, Clark, 1986; Roitt, 1991).In both the heavy chain and the light chain variable regions of IgGimmunoglobulins, there are four framework regions (FR1 through FR4)separated respectively by three complementarity determining regions(CDR1 through CDR3). The framework regions (FRs) maintain the tertiarystructure of the paratope, which is the portion of the antibody which isinvolved in the interaction with the antigen. The CDRs, and inparticular the CDR3 regions, and more particularly the heavy chain CDR3contribute to antibody specificity. Because these CDR regions and inparticular the CDR3 region confer antigen specificity on the antibodythese regions may be incorporated into other antibodies or peptides toconfer the identical specificity onto that antibody or peptide.

According to one embodiment, the peptide of the invention is an intactsoluble monoclonal antibody in an isolated form or in a pharmaceuticalpreparation. An intact soluble monoclonal antibody, as is well known inthe art, is an assembly of polypeptide chains linked by disulfidebridges. Two principle polypeptide chains, referred to as the lightchain and heavy chain, make up all major structural classes (isotypes)of antibody. Both heavy chains and light chains are further divided intosubregions referred to as variable regions and constant regions. As usedherein the term “monoclonal antibody” refers to a homogenous populationof immunoglobulins which specifically bind to an epitope (i.e. antigenicdeterminant), e.g., of MHC class II HLA-DR.

The peptide useful according to the methods of the present invention maybe an intact humanized a monoclonal antibody. A “humanized monoclonalantibody” as used herein is a human monoclonal antibody or functionallyactive fragment thereof having human constant regions and a binding CDR3region from a mammal of a species other than a human. Humanizedmonoclonal antibodies may be made by any method known in the art.Humanized monoclonal antibodies, for example, may be constructed byreplacing the non-CDR regions of a non-human mammalian antibody withsimilar regions of human antibodies while retaining the epitopicspecificity of the original antibody. For example, non-human CDRs andoptionally some of the framework regions may be covalently joined tohuman FR and/or Fc/pFc′ regions to produce a functional antibody. Thereare entities in the United States which will synthesize humanizedantibodies from specific murine antibody regions commercially, such asProtein Design Labs (Mountain View Calif.). For instance, a humanizedform of the Pharmingen anti-Fas antibody used in the attached Examplescould be easily prepared and used according to the methods of theinvention.

European Patent Application 0239400, the entire contents of which ishereby incorporated by reference, provides an exemplary teaching of theproduction and use of humanized monoclonal antibodies in which at leastthe CDR portion of a murine (or other non-human mammal) antibody isincluded in the humanized antibody. Briefly, the following methods areuseful for constructing a humanized CDR monoclonal antibody including atleast a portion of a mouse CDR. A first replicable expression vectorincluding a suitable promoter operably linked to a DNA sequence encodingat least a variable domain of an Ig heavy or light chain and thevariable domain comprising framework regions from a human antibody and aCDR region of a murine antibody is prepared. Optionally a secondreplicable expression vector is prepared which includes a suitablepromoter operably linked to a DNA sequence encoding at least thevariable domain of a complementary human Ig light or heavy chainrespectively. A cell line is then transformed with the vectors.Preferably the cell line is an immortalized mammalian cell line oflymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cellline, or is a normal lymphoid cell which has been immortalized bytransformation with a virus. The transformed cell line is then culturedunder conditions known to those of skill in the art to produce thehumanized antibody.

As set forth in European Patent Application 0239400 several techniquesare well known in the art for creating the particular antibody domainsto be inserted into the replicable vector. (Preferred vectors andrecombinant techniques are discussed in greater detail below.) Forexample, the DNA sequence encoding the domain may be prepared byoligonucleotide synthesis. Alternatively a synthetic gene lacking theCDR regions in which four framework regions are fused together withsuitable restriction sites at the junctions, such that double strandedsynthetic or restricted subcloned CDR cassettes with sticky ends couldbe ligated at the junctions of the framework regions. Another methodinvolves the preparation of the DNA sequence encoding the variable CDRcontaining domain by oligonucleotide site-directed mutagenesis. Each ofthese methods is well known in the art. Therefore, those skilled in theart may construct humanized antibodies containing a murine CDR regionwithout destroying the specificity of the antibody for its epitope.

Human monoclonal antibodies may be made by any of the methods known inthe art, such as those disclosed in U.S. Pat. No. 5,567,610, issued toBorrebaeck et al., U.S. Pat. No. 565,354, issued to Ostberg, U.S. Pat.No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001(1984), Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), and Boerneret al., J. Immunol., 147: 86-95 (1991). In addition to the conventionalmethods for preparing human monoclonal antibodies, such antibodies mayalso be prepared by immunizing transgenic animals that are capable ofproducing human antibodies (e.g., Jakobovits et-al., PNAS USA, 90: 2551(1993), Jakobovits et al., Nature, 362: 255-258 (1993), Bruggermann etal., Year in Immuno., 7:33 (1993) and U.S. Pat. No. 5,569,825 issued toLonberg).

The binding peptides may also be functionally active antibody fragments.Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions of theantibody, for example, are effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically cleaved, or which has been produced without thepFc′ region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. An isolated F(ab′)₂ fragment isreferred to as a bivalent monoclonal fragment because of its two antigenbinding sites. Similarly, an antibody from which the Fc region has beenenzymatically cleaved, or which has been produced without the Fc region,designated an Fab fragment, retains one of the antigen binding sites ofan intact antibody molecule. Proceeding further, Fab fragments consistof a covalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd (heavy chain variable region). The Fd fragmentsare the major determinant of antibody specificity (a single Fd fragmentmay be associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

The terms Fab, Fc, pFc′, F(ab′)₂ and Fv are used consistently with theirstandard immunological meanings [Klein, Immunology (John Wiley, NewYork, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations ofModern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991)Essential Immunology, 7th Ed., (Blackwell Scientific Publications,Oxford)].

The B7 and UCP expression vectors and other relevant expression vectorsdescribed herein can be prepared and inserted into cells using routineprocedures known in the art. These procedures are set forth below inmore detail. The term “IRM” (immune recognition molecule) nucleic acidis used herein to refer to each of the nucleic acids encompassed by theexpression vectors described herein. Although UCP is not an immunemolecule the term IRM is used to encompass UCP nucleic acids to simplifythe discussion. “IRM nucleic acid”, as used herein, refers to a nucleicacid molecule which: (1) hybridizes under stringent conditions to anucleic acid having the sequence of SEQ ID NO:1, 3, 5, 7, 9, and 11 and(2) codes for a IRM polypeptide (i.e., the respective-immune recognitionpolypeptide). The preferred IRM nucleic acid has the nucleic acidsequence of SEQ ID NO:1, 3, 5, 7, 9, and 11 (the nucleic acids encodingthe human B7.1, B7.2, UCP-1, UCP-2, UCP-3S, and CD28 polypeptidesrespectively). The IRM nucleic acids may be intact IRM nucleic acidswhich include the nucleic acid sequence of Sequence ID No. 1-5 as wellas homologs and alleles of a nucleic acid having the sequence of SEQ IDNO:1, 3, 5, 7, 9, and 11. Intact IRM nucleic acids further embracenucleic acid molecules which differ from the sequence of SEQ ID NO:1, 3,5, 7, 9, and 11 in codon sequence due to the degeneracy of the geneticcode. The IRM nucleic acids of the invention may also be functionallyequivalent variants, analogs and fragments of the foregoing nucleicacids. “Functionally equivalent”, in reference to a IRM nucleic acidvariant, analog or fragment, refers to a nucleic acid that codes for aIRM polypeptide that is capable of functioning as an immune recognitionmolecule or an uncoupling protein. The invention further embracescomplements of the foregoing nucleic acids or of unique fragments of theforegoing nucleic acids. Such complements can be used, for example, asantisense nucleic acids for inhibiting the expression of IRM in a cellin order to create an experimental model of a cell in which IRM is notexpressed.

The IRM nucleic acid molecules can be identified by conventionaltechniques, e.g., by identifying nucleic acid sequences which code forIRM polypeptides and which hybridize to a nucleic acid molecule havingthe sequence of SEQ ID NO:1, 3, 5, 7, 9, and 11 under stringentconditions. The term “stringent conditions”, as used herein, refers toparameters with which the art is familiar. More specifically, stringentconditions, as used herein, refer to hybridization at 65° C. inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone,0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA).SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS is sodiumdodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid. Afterhybridization, the membrane to which the DNA is transferred is washed at2×SSC at room temperature and then at 0.1×SSC/0.1×SDS at 65° C.

There are other conditions, reagents, and so forth which can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions and, thus, they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of the IRM nucleic acid of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for the expression of molecules, such as IRM, whichcan be isolated, followed by purification and sequencing of thepertinent nucleic acid molecule. In screening for IRM nucleic acidsequences, a Southern blot may be performed using the foregoingconditions, together with a radioactive probe. After washing themembrane to which the DNA is finally transferred, the membrane can beplaced against x-ray film to detect the radioactive signal.

In general, homologs and alleles typically will share at least 40%nucleotide identity with SEQ ID NO:1, 3, 5, 7, 9, and 11; in someinstances, will share at least 50% nucleotide identity; and in stillother instances, will share at least 60% nucleotide identity. Thepreferred homologs have at least 70% sequence homology to SEQ ID NO:1,3, 5, 7, 9, and 11. More preferably the preferred homologs have at least80% and, most preferably, at least 90% sequence homology to SEQ ID NO:1,3, 5, 7, 9, and 11-5.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the naturally occurring nucleicacid that codes for the human IRM polypeptide. For example, serineresidues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Eachof the six codons is equivalent for the purposes of encoding a serineresidue. Thus, it will be apparent to one of ordinary skill in the artthat any of the serine-encoding nucleotide codons may be employed todirect the protein synthesis apparatus, in vitro or in vivo, toincorporate a serine residue. Similarly, nucleotide sequence tripletswhich encode other amino acid residues include, but are not limited to,CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG(arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC and ATT (isoleucine codons). Otheramino acid residues may be encoded similarly by multiple nucleotidesequences. Thus, the invention embraces degenerate nucleic acids thatdiffer from the naturally occurring nucleic acids in codon sequence dueto the degeneracy of the genetic code.

The IRM nucleic acid, in one embodiment, is operably linked to a geneexpression sequence which directs the expression of the IRM nucleic acidwithin a eukaryotic cell. The “gene expression sequence” is anyregulatory nucleotide sequence, such as a promoter sequence orpromoter-enhancer combination, which facilitates the efficienttranscription and translation of the IRM nucleic acid to which it isoperably linked. The gene expression sequence may, for example, be amammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, and β-actin.Exemplary viral promoters which function constitutively in eukaryoticcells include, for example, promoters from the simian virus, papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcomavirus, cytomegalovirus, the long terminal repeats (LTR) of moloneyleukemia virus and other retroviruses, and the thymidine kinase promoterof herpes simplex virus. Other constitutive promoters are known to thoseof ordinary skill in the art. The promoters useful as gene expressionsequences of the invention also include inducible promoters. Induciblepromoters are expressed in the presence of an inducing agent. Forexample, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined IRM nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

Preferably, the IRM nucleic acid of the invention is linked to a geneexpression sequence which permits expression of the IRM nucleic acid inthe local environment of a cell, e.g. a damaged nerve cell. In someembodiments the gene expression sequence permits expression of the IRMnucleic acid in a human nerve cell or a neural activating cell. Asequence which permits expression of the IRM nucleic acid in a nervecell or a neural activating cell is one which is selectively active innerve cell or a neural activating cell and thereby causes the expressionof the IRM nucleic acid in these cells. Those of ordinary skill in theart will be able to easily identify promoters that are capable ofexpressing a IRM nucleic acid in a nerve cell or a neural activatingcell, as well as other known cells.

The IRM nucleic acid sequence and the gene expression sequence are saidto be “operably linked” when they are covalently linked in such a way asto place the transcription and/or translation of the IRM coding sequenceunder the influence or control of the gene expression sequence. If it isdesired that the IRM sequence be translated into a functional protein,two DNA sequences are said to be operably linked if induction of apromoter in the 5′ gene expression sequence results in the transcriptionof the IRM sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the IRM sequence, or (3) interfere with theability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to aIRM nucleic acid sequence if the gene expression sequence were capableof effecting transcription of that IRM nucleic acid sequence such thatthe resulting transcript might be translated into the desired protein orpolypeptide.

The IRM nucleic acid of the invention can be delivered to the cell aloneor in association with a vector. In its broadest sense, a “vector” isany vehicle capable of facilitating: (1) delivery of a IRM molecule to atarget cell or (2) uptake of a IRM molecule by a target cell.Preferably, the vectors transport the IRM molecule into the target cellwith reduced degradation relative to the extent of degradation thatwould result in the absence of the vector. Optionally, a “targetingligand” can be attached to the vector to selectively deliver the vectorto a cell which expresses on its surface the cognate receptor for thetargeting ligand. In this manner, the vector (containing a IRM nucleicacid) can be selectively delivered to a cell in, e.g., an injured nervetissue. In general, the vectors useful in the invention are divided intotwo classes: biological vectors and chemical/physical vectors.Biological vectors are useful for delivery/uptake of IRM nucleic acidsto/by a target cell. Chemical/physical vectors are also useful fordelivery/uptake of IRM nucleic acids to/by a target cell.

Biological vectors include, but are not limited to, plasmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the nucleicacid sequences of the invention, and free nucleic acid fragments whichcan be attached to the nucleic acid sequences of the invention. Viralvectors are a preferred type of biological vector and include, but arenot limited to, nucleic acid sequences from the following viruses:retroviruses, such as: Moloney murine leukemia virus; Harvey murinesarcoma virus; murine mammary tumor virus; Rous sarcoma virus;adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses;Epstein-Barr viruses; papilloma viruses; herpes viruses; vacciniaviruses; polio viruses; and RNA viruses such as any retrovirus. One canreadily employ other vectors not named but known in the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. In general, theretroviruses are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual,” W.H.Freeman Co., New York (1990) and Murry, E. J. Ed. “Methods in MolecularBiology,” vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus can beengineered to be replication-deficient and is capable of infecting awide range of cell types and species. It further has advantages, such asheat and lipid solvent stability; high transduction frequencies in cellsof diverse lineages; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

In addition to the biological vectors, chemical/physical vectors may beused to deliver a IRM molecule to a target cell and facilitate uptakethereby. As used herein, a “chemical/physical vector” refers to anatural or synthetic molecule, other than those derived frombacteriological or viral sources, capable of delivering the IRM moleculeto a cell.

A preferred chemical/physical vector of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0 μm can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., (1981) 6:77). In order for aliposome to bean efficient gene transfer vector, one or more of thefollowing characteristics should be present: (1) encapsulation of thegene of interest at high efficiency with retention of biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information.

Liposomes may be targeted to a particular tissue, such as the site of atumor, by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein. Ligands which may beuseful for targeting a liposome to a tumor cell include, but are notlimited to: intact or fragments of IRM which interact with tumor cellspecific receptor and molecules which interact with the cell surfacemarkers of tumor cells such as antibodies. Such ligands may easily beidentified by binding assays well known to those of skill in the art.Additionally, the vector may be coupled to a nuclear targeting peptide,which will direct the IRM nucleic acid to the nucleus of the host cell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, (1985) 3:235-241.

In one particular embodiment, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, claiming priority to U.S. patentapplication Ser. No. 213,668, filed Mar. 15, 1994). PCT/US/0307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining an exogenous gene under the control of an appropriatepromotor. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instantinvention, the IRM nucleic acids described herein are encapsulated ordispersed within the biocompatible, preferably biodegradable polymericmatrix disclosed in PCT/US/03307.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein the IRM molecule is dispersed throughout asolid polymeric matrix) or a microcapsule (wherein the IRM molecule isstored in the core of a polymeric shell). Other forms of the polymericmatrix for containing the IRM molecule include films, coatings, gels,implants, and stents. The size and composition of the polymeric matrixdevice is selected to result in favorable release kinetics in the tissueinto which the matrix is introduced. The size of the polymeric matrixfurther is selected according to the method of delivery which is to beused, typically injection into a tissue or administration of asuspension by aerosol into the nasal and/or pulmonary areas. Preferablywhen an aerosol route is used the polymeric matrix and IRM molecule areencompassed in a surfactant vehicle. The polymeric matrix compositioncan be selected to have both favorable degradation rates and also to beformed of a material which is bioadhesive, to further increase theeffectiveness of transfer when the matrix is administered to a nasaland/or pulmonary surface that has sustained an injury. The matrixcomposition also can be selected not to degrade, but rather, to releaseby diffusion over an extended period of time.

In another embodiment the chemical/physical vector is a biocompatiblemicrosphere that is suitable for oral delivery. Such microspheres aredisclosed in Chickering et al., Biotech. And Bioeng., (1996) 52:96-101and Mathiowitz et al., Nature, (1997) 386:410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the IRM nucleic acids of the invention to the subject.Biodegradable matrices are preferred. Such polymers may be natural orsynthetic polymers. Synthetic polymers are preferred. The polymer isselected based on the period of time over which release is desired,generally in the order of a few hours to a year or longer. Typically,release over a period ranging from between a few hours and three totwelve months is most desirable. The polymer optionally is in the formof a hydrogel that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

In general, the IRM nucleic acids are delivered using a bioerodibleimplant by way of diffusion, or more preferably, by degradation of thepolymeric matrix. Exemplary synthetic polymers which can be used to formthe biodegradable delivery system include: polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene,polyvinylpyrrolidone, and polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Examples of non-biodegradable; polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

Compaction agents also can be used alone, or in combination with, abiological or chemical/physical vector of the invention. A “compactionagent”, as used herein, refers to an agent, such as a histone, thatneutralizes the negative charges on the nucleic acid and thereby permitscompaction of the nucleic acid into a fine granule. Compaction of thenucleic acid facilitates the uptake of the nucleic acid by the targetcell. The compaction agents can be used alone, i.e., to deliver the IRMmolecule in a form that is more efficiently taken up by the cell or,more preferably, in combination with one or more of the above-describedvectors.

Other exemplary compositions that can be used to facilitate uptake by atarget cell of the IRM nucleic acids include calcium phosphate and otherchemical mediators of intracellular transport, microinjectioncompositions, electroporation and homologous recombination compositions(e.g., for integrating a IRM nucleic acid into a preselected locationwithin the target cell chromosome).

In addition to the expression vectors, the invention also encompassesthe use of antisense oligonucleotides that selectively bind to a IRMnucleic acid molecule, and dominant negative IRM to reduce theexpression of IRM. Antisense oligonucleotides are useful, for example,for preparing an animal model of a subject having a neurodegenerativedisorder. Such animal models can be used in screening assays foridentifying therapeutic drugs for treating neurodegenerative disorders.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide which hybridizes under physiologicalconditions to DNA comprising a particular gene or to an RNA transcriptof that gene and, thereby, inhibits the transcription of that geneand/or the translation of the mRNA. The antisense molecules are designedso as to hybridize with the target gene or target gene product andthereby, interfere with transcription or translation of the targetmammalian cell gene. Those skilled in the art will recognize that theexact length of the antisense oligonucleotide and its degree ofcomplementarity with its target will depend upon the specific targetselected, including the sequence of the target and the particular baseswhich comprise that sequence. The antisense must be a unique fragment. Aunique fragment is one that is a ‘signature’ for the larger nucleicacid. It, for example, is long enough to assure that its precisesequence is not found in molecules outside of the IRM gene. As will berecognized by those skilled in the art, the size of the unique fragmentwill depend upon its conservancy in the genetic code. Thus, some regionsof SEQ ID NO:1, 3, 5, 7, 9, and 11, will require longer segments to beunique while others will require only short segments, typically between12 and 32 base pairs (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases long).

It is preferred that the antisense oligonucleotide be constructed andarranged so as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the known sequence of a gene that is targeted forinhibition by antisense hybridization, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 7 and, more preferably, atleast 15 consecutive bases which are complementary to the target. Mostpreferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or RNA (e.g., mRNA) transcripts,in preferred embodiments the antisense oligonucleotides arecomplementary to 5′ sites, such as translation initiation, transcriptioninitiation or promoter sites, that are upstream of the gene that istargeted for inhibition by the antisense oligonucleotides. In addition,3′-untranslated regions may be targeted. Furthermore, 5′ or 3′ enhancersmay be targeted. Targeting to mRNA splice sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In at least some embodiments, the antisense is targeted, preferably, tosites in which mRNA secondary structure is not expected (see, e.g.,Sainio et al., Cell Mol. Neurobiol., (1994) 14(5):439-457) and at whichproteins are not expected to bind. The selective binding of theantisense oligonucleotide to a mammalian target cell nucleic acideffectively decreases or eliminates the transcription or translation ofthe mammalian target cell nucleic acid molecule. Reduction intranscription or translation of the nucleic acid molecule is desirablein preparing an animal model for further defining the role played by themammalian target cell nucleic acid in modulating an adverse medicalcondition.

The invention also includes the use of a “dominant negative UCP”polypeptide. A dominant negative polypeptide is an inactive variant of aprotein, which, by interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery orcompetes with the active protein, thereby reducing the effect of theactive protein. For example, a dominant negative receptor which binds aligand but does not transmit a signal in response to binding of theligand can reduce the biological effect of expression of the ligand.Likewise, a dominant negative catalytically-inactive kinase whichinteracts normally with target proteins but does not phosphorylate thetarget proteins can reduce phosphorylation of the target proteins inresponse to a cellular signal. Similarly, a dominant negativetranscription factor which binds to a promoter site in the controlregion of a gene but does not increase gene transcription can reduce theeffect of a normal transcription factor by occupying promoter bindingsites without increasing transcription.

The end result of the expression of a dominant negative polypeptide asused herein in a cell is a reduction in function of active UCP. One ofordinary skill in the art can assess the potential for a dominantnegative variant of a protein, and using standard mutagenesis techniquesto create one or more dominant negative variant polypeptides. Forexample, one of ordinary skill in the art can modify the sequence of theUCP by site-specific mutagenesis, scanning mutagenesis, partial genedeletion or truncation, and the like. See, e.g., U.S. Pat. No. 5,580,723and Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisanthen can test the population of mutagenized polypeptides for diminutionin a selected and/or for retention of such an activity. Other similarmethods for creating and testing dominant negative variants of a proteinwill be apparent to one of ordinary skill in the art.

In other aspects the invention includes transgenic animals and cellstransfected with the IRM's. Additionally, complements of the IRM nucleicacids described above can be useful as anti-sense oligonucleotides,e.g., by delivering the anti-sense oligonucleotide to an animal toinduce a “knockout” phenotype. The administration of anti-sense RNAprobes to block gene expression is discussed in Lichtenstein, C., Nature333:801-802 (1988).

Alternatively, the IRM nucleic acids can be used to prepare a non-humantransgenic animal. A “transgenic animal” is an animal having cells thatcontain DNA which has been artificially inserted into a cell, which DNAbecomes part of the genome of the animal which develops from that cell.Preferred transgenic animals are primates, mice, rats, cows, pigs,horses, goats, sheep, dogs and cats. Animals suitable for transgenicexperiments can be obtained from standard commercial sources such asCharles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), HarlanSprague Dawley (Indianapolis, Ind.), etc. Transgenic animals having aparticular property associated with a particular disease can be used tostudy the affects of a variety of drugs and treatment methods on thedisease, and thus serve as genetic models for the study of a number ofhuman diseases. The invention, therefore, contemplates the use of IRMknockout and transgenic animals as models for the study ofneurodegenerative disorders.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division.See e.g., Brinster et al., Proc. Nat. Acad. Sci. USA, 82: 4438 (1985);Brinster et al., cell 27: 223 (1981); Costantini et al., Nature 294: 982(1981); Harpers et al., Nature 293: 540 (1981); Wagner et al., Proc.Nat. Acad. Sci. USA 78:5016 (1981); Gordon et al., Proc. Nat. Acad. Sci.USA 73: 1260 (1976). The fertilized egg is then implanted into theuterus of the recipient female and allowed to develop into an animal.

An alternative method for producing transgenic animals involves theincorporation of the desired gene sequence into a virus which is capableof affecting the cells of a host animal. See e.g., Elbrecht et al.,Molec. Cell. Biol. 7: 1276 (1987); Lacey et al., Nature 322: 609 (1986);Leopol et al., Cell 51: 885 (1987). Embryos can be infected withviruses, especially retroviruses, modified to carry the nucleotidesequences which encode IRM proteins or sequences which disrupt thenative IRM gene to produce a knockout animal.

Another method for producing transgenic animals involves the injectionof pluripotent embryonic stem cells into a blastocyst of a developingembryo. Pluripotent stem cells derived from the inner cell mass of theembryo and stabilized in culture can be manipulated in culture toincorporate nucleotide sequences of the invention. A transgenic animalcan be produced from such cells through implantation into a blastocystthat is implanted into a foster mother and allowed to come to term. Seee.g., Robertson et al., Cold Spring Harbor Conference Cell Proliferation10: 647 (1983); Bradley et al., Nature 309: 255 (1984); Wagner et al.,Cold Spring Harbor Symposium Quantitative Biology 50: 691 (1985).

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia, 47: 897-905 (1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No. 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. Females are placed with males, and the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer etal., Cell, 63:1099-1112 (1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press (1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination. Capecchi,Science 244: 1288-1292 (1989). Methods for positive selection of therecombination event (e.g., neo resistance) and dual positive-negativeselection (e.g., neo resistance and gangcyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al., Nature, 338: 153-156(1989). The final phase of the procedure is to inject targeted ES cellsinto blastocysts and to transfer the blastocysts into pseudopregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene.

Procedures for the production of non-rodent mammals and other animalshave been discussed by others. See Houdebine and Chourrout, supra;Pursel et al., Science 244: 1281-1288 (1989); and Simms et al.,Bio/Technology, 6: 179-183 (1988).

Inactivation or replacement of the endogenous IRM genes can be achievedby a homologous recombination system using embryonic stem cells. Theresultant transgenic non-human mammals having a knockout characteristicmay be used as a model for neurodegenerative disorders. Nerve cellswhich do not express IRMs may be predisposed to apoptosis and unable todifferentiate and thus, produce a neurodegenerative phenotype. A varietyof therapeutic drugs can be administered to the phenotypicallyneurodegenerative animals to determine the affect of the therapeuticdrugs on nerve cell differentiation. In this manner, therapeutic drugswhich are useful for preventing or reducing neurodegenerative disorderscan be identified. Such agents are useful for, e.g., treating spinalchord injuries or Parkinson's disease.

Additionally, a normal or mutant version of IRM can be inserted into themouse germ line to produce transgenic animals which constitutively orinducible express the normal or mutant form of IRM. These animals areuseful in studies to define the role and function of IRM in cells.

The metabolic modifying agent, apoptotic chemotherapeutic agent, MHCclass II HLA-DR inducing agent, MHC class II HLA-DR ligand, B7 receptorblocking agent, B7 inducing agent, and B7 receptor inducing agentdescribed herein are commercially available compounds, are derived fromcommercially available compounds or are synthesized de novo usingroutine chemical synthetic procedures known to those of ordinary skillin the art.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts. As used herein, a composition of anmetabolic modifying agent and an apoptotic chemotherapeutic agent meansthe compounds described above as well as salts thereof and a compositionof an MHC class II HLA-DR inducing agent and an MHC class II HLA-DRligand means the compounds described above as well as salts thereof.

The compositions of the invention may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human or other animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the molecules of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the compositions of theinvention, which is preferably isotonic with the blood of the recipient.This aqueous preparation may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation also may be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing thecompositions of the invention into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing thecompositions of the invention into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the compositions of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the compositions of the invention is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not to be construed aslimiting the present invention to these examples. As will be apparent toone of ordinary skill in the art, the present invention will findapplication in a variety of compositions and methods.

EXAMPLES Example 1 Metabolic State of a Cell is Indicative of CellSurface Fas Expression and Sensitivity/Resistance to Cell Death

1. Resistance to apoptosis is characterized by failure to express Fas:The cell lines utilized herein include L1210, a leukemic cell line;HL60, a human pro-myelocytic cell line; and PC12, a pheochromocytomacell line which can be induced to differentiate into a neuronal cellline in the presence of NGF (Lindenboim, L, et al., Cancer Res, 1995,55:1242-7). Each cell line was examined in parallel with apoptoticresistant sublines: L1210 DDP, HL60 MDR, and PC12Trk. L1210 DDP areresistant to cisplatin and methotrexate; HL60 MDR are resistant toadriamycin induced apoptosis; PC12 TrkA, which have been transfectedwith TrkA which results in constitutively expression the NGF receptors,are not susceptible to alcohol and NGF withdrawal as are the PC12 cells.

The apoptosis sensitive cells from each tissue origin weremorphologically round, non-adherent, rapidly dividing cells, with theexception of the PC12 cell line. The apoptosis resistant cells from alltissue origins were morphologically large, adherent, and slowly dividingcells.

The recently characterized molecules, Fas (CD95) and Fas Ligand (CD95L),have been strongly implicated in the process of apoptotic death (Muller,M, et al., J Clin Invest, 1997, 99:403-413). We examined expression ofFas on the above-identified cell lines. Independent of tissue origin,all of the apoptosis resistant lines fail to express cell surface Fasboth constitutively and in the presence of agents that induce apoptosisin the parental cell lines, as shown in FIG. 1A.

FIG. 1A shows a flow cytometric analysis of Fas expression. Isotypecontrol (thick line) versus FITC-anti-Fas (Pharmingen) (thin lines), on(from top to bottom) L1210; PC12; and HL60 cells, left panels asindicated. Panels on the right are staining of resistant cell linesL1210DDP, PC12Trk; and HL60MDR. The histograms representing isotypecontrol (thick) versus FITC-anti-Fas (thin lines) are completelyoverlapping on the right panels, indicating an absence of Fasexpression. A Coulter Epics Elite flow cytometer with a singleexcitation wavelength (488 nm) and band filters for PE (575 nm), FITC(525 nm) and Red613 (613 nm) was used to analyze the stained cells. Eachsample population was classified for cell size (forward scatter) andcomplexity (side scatter), gated on a population of interest andevaluated using 40,000 cells. Criteria for positive staining wereestablished by comparison with the intensity of the isotype controls,thick lines.

FIG. 1B is a flow cytometric analysis of extracellular Fas andintracellular UCP and bcl-2 expression. Isotype control (Thick line)versus FITC-anti-Fas (Pharmingen) (Thin lines), on L1210, left panels,and L1210DDP, right panels. The histograms represent Isotype control(thin) versus FITC-anti-Fas (thick lines). A coulter Epics Elite flowcytometer with a single excitation wavelength (488 nm) and band filtersfor PE (575 nm), FITC (525 nm) and Red613 (613 nm) was used to analyzethe stained cells. Each sample population was classified for cell size(forward scatter) and complexity (side scatter), gated on a populationof interest and evaluated using 40,000 cells. Criteria for positivestaining were established by comparison with Isotype controls, thinlines to specific stain, thick lines.

2. Resistance to apoptosis is characterized by relatively high rates ofglucose oxidation and utilization: We performed experiments to examinethe correlation between cell surface Fas expression and glucosemetabolism. As a prototype for the Fas positive and Fas negative cellswe used the L1210 and the L1210DDP cell lines, as Fas positive and Fasnegative, respectively. We directly measured the rates of glucoseutilization and oxidation of L1210 and L1210DDP. Ratios were generatedby using nanomolar values.

Rate of glucose utilization was measured by the method of Ashcroft etal. Briefly, cells were incubated 90 min at 37° C. in 100 μl KRB,glucose (5.5 mM), 1.3 μCi D-[5-³H] glucose (Amersham, Arlington Heights,Ill.). The reaction was carried out in a 1 ml cup contained in a rubberstoppered 20 ml scintillation vial that had 500 μl of distilled watersurrounding the cup. Glucose metabolism was stopped by injecting 100 μl1 M Hcl through the stopper into the cup. An overnight incubation at 37°C. was carried out to allow equilibration of the [³H]-H₂O in thereaction cup and the distilled water, followed by liquid scintillationcounting of the distilled water.

Rate of glucose oxidation was measured by incubating cells for 90 min at37° C. in 100 ml of reaction buffer, glucose (2.8, 8.3, 27.7 mmol/l),1.7 mCi (U-14C glucose). The reaction was carried out in a 1 ml cup in a20 ml scintillation vial capped by a rubber stopper with a center wellthat contains filter paper. Metabolism was stopped and CO₂ liberatedwith 300 ml 1 mol/l HCl injected through the stopper into the cupcontaining the cells. CO₂ was trapped in the filter paper by injecting10 ml 1 mol/l KOH into the center well, followed 2 hours later by liquidscintillation counting. Tubes containing NaHCO₃ and no cells were usedto estimate the recovery of ¹⁴CO₂ in the filter paper, routinely closeto 100%.

The results are presented in Table 1.

TABLE 1 Glucose Metabolism in L1210/0 and L1210/DDP L1210/0 L1210/DDPGlucose Utilization 1740 ± 920 3470 ± 460  (pmol glucose/90 min/50,000cells) Glucose Oxidation 235 ± 7  428 ± 124 (pmol glucose/90 min/50,000cells) Glucose Utilization/Oxidation 7.4 8.1

Because the L1210 and L1210DDP cells are tumor cell lines and are likelyto have increased ratios of glucose oxidation to utilization (Warburg,0, et al., Klin Woch, 1926, 5:829-832), we measured glucose utilizationon normal lymphocytes. We isolated 10⁶ splenic lymphocytes from C57BL/6animals, Fas-deficient C57BL/6 (B6.lpr), and FasL defective C57BL/6(B6.gld) animals. The rate of glucose utilization and oxidation of theFas deficient and the FasL deficient lymphocytes are demonstrated inTable 2. The ratio of glucose utilization to oxidation is highest in lprlymphocytes and lowest in wild type normal, quiescent lymphocytes.

TABLE 2 Glucose Metabolism in Lymphocytes from Normal, Fas Deficient andFasL Deficient Mice b6 lpr gld GLUCOSE UTILIZATION (nmol glucose/90mins/50,000 cells) 0.04 0.36 0.22 GLUCOSE OXIDATION (pmol glucose/90mins/50,000 cells) 73.24 164.51 122.82 CELL TYPE RATIO GLUCOSEUTILIZATION/ b6 0.55 GLUCOSE OXIDATION lpr 2.19 gld 1.79

These data (Table 1 & 2) demonstrate high rates of glucose utilizationand oxidation of both tumor lines relative to the normal lymphocytes;and higher rates of glucose utilization and oxidation of the apoptoticresistant line relative to the wild type. There is an importantdifference in the ratio of glucose utilization to oxidation betweennormal and Fas or FasL deficient animals, with the ratio being higherfor lymphocytes from both mutant strains of animals. The consequences ofuncoupling are a decrease in mitochondrial membrane potential; use offat as a carbon source increased rate of glycolysis, increased rate ofelectron transport, and energy dissipation, in a form other than ATP.These data suggest that there is an increase in proton leak in the cellswith high rates of glucose oxidation and utilization relative to thenormal cells, suggesting some degree of uncoupling may have occurred inthese cells.

3. Fas Expression Increases as a Function of Glucose: We investigatedthe effect of increasing concentrations of glucose on cell surface Fasexpression. L1210 and L1210/DDP cells were cultured in glucose free RPMImedia or in media supplemented with insulin and glucose for 16 hours.Intra- and extracellular Fas expression was determined by labeling thecells with FITC-conjugated anti-Fas antibodies (Pharmingen), orFITC-conjugated isotype control, then subtracting the fluorescenceintensity of the isotype staining from Fas staining for each treatmentgroup.

These data show that Fas expression increases as a function of glucoseconcentration, FIG. 2, and that as a result the cell surface Fasnegative L1210/DDP begin to express cell surface Fas.

L1210, upper panels, and L1210/DDP cells, lower panels, were cultured inglucose free RPMI media, filled circles, or in media supplemented withinsulin and glucose, squares, for 16 hours. Intra-and extracellular Fasexpression was determined by labeling the cells with FITC-conjugatedanti-Fas antibodies, or FITC-conjugated isotype control, thensubtracting the fluorescence intensity of the isotype staining from Fasstaining for each treatment group (FIG. 2). Results are presented asgeometric mean fluorescence over background.

4. Treatment of L1210 DDP cells with staurosporin restores Fasexpression and susceptibility to drug-induced apoptosis: L1210, but notL1210 DDP, undergo apoptotic cell death. We treated L1210 or L1210 DDPcells with the staurosporin, which inhibits protein kinase C andincreases mitochondrial membrane potential, or an anti-cancer agent towhich both cells are sensitive, adriamycin. Fas expression was increasedor induced on both L1210 and L1210 DDP, respectively, in the presence ofstaurosporin or adriamycin (FIG. 3). The L1210 DDP changedmorphologically and began to divide rapidly, changes which appeared tocorrespond with a reversion back to the phenotype of the L1210 cells.These results demonstrate that Fas expression results in parallel withaltered metabolic activity.

FIG. 3 shows the treatment of L1210 DDP cells with staurosporin restoresFas expression and susceptibility to drug-induced apoptosis.

5. Confocal microscopy reveals that resistance to apoptosis ischaracterized by intra-(but not extra) cellular Fas expression: L1210DDP cells express no cell surface Fas. To address the possibility thatFas is expressed, but has been targeted to a subcellular organelle, wepermeabilized and stained L1210 and L1210DDP cells with fluorochromeconjugated anti-Fas antibody (J02.2, Pharmingen). The cells wereexamined by confocal microscopy. (This experiment was representative offour experiments).

Our data indicate that L1210 DDP cells express Fas in an intracellular,cytosolic compartment. Fluorochrome-conjugated isotype matched antibodywas used as control. Additionally, these data also demonstrate that theFas negative, apoptosis resistant cells, express intracellular Fas.

6. Fas-deficient (lpr) lymphocytes express intra-(but not extra-)cellular Fas molecules: We isolated lymphocytes from spleens of C57BL/6mice and from C57BL6 transgenics having the lpr mutation (loss of Fassensitivity). Cells were stained with fluorescein conjugated hamsteranti-Fas and examined by confocal microscopy.

Results demonstrate that unstimulated, non-permeabilized splenocytesfrom C57BL/6 animals express Fas at low levels relative to isotypecontrols. Interestingly, significant levels of Fas expression weredetected in permeabilized normal lymphocytes. As expected,non-permeabilized cells from C57BL6.lpr animals express no detectablecell surface Fas relative to isotype control. Interestingly,intracellular Fas staining of permeabilized splenocytes from C57B1/6.lpranimals reveals intracellular expression of Fas. These resultsdemonstrate that mutations affecting susceptibility to Fas-induced deathprevent cell surface, but not intracellular expression of the Fasmolecule.

7. Anti-cancer agents induce susceptibility to Fas-induced cell death:To determine if the anti-cancer agent methotrexate sensitizes L1210 orL1210/DDP cells to Fas induced cell death, we cultured L1210 cells inthe presence or absence of 10⁻⁸ M methotrexate for 72 hours. Each groupof cells was cultured on uncoated plates or plates coated with 10 g/mlanti-Fas (Jo.2.2, Pharmingen). We analyzed cell death using flowcytometry. Forward angle and 90 degree light scatter were used todistinguish between live and dead cells. Dead cells were gated asforward angle light scatter low/high ethidium bromide retaining cells.Percent death was calculated over the total number of cells acquired. InTable 3 below, values indicate % dead cells over background of untreatedcells.

TABLE 3 Fas-induced cell death L1210/0 L1210/DDP Control 4.72 40.88anti-Fas Coated Plates 79.98 46.60

Additionally, L1210 and L1210/DDP cells were treated with 10-8 Mmethotrexate for 24 hours. Flow cytometric analysis revealed twopopulations based on forward side scatter. The forward scatter highpopulations did not take ethidium bromide and were therefore viable. Theforward scatter low populations took up ethidium bromide differentially.The L1210 cells took up a moderate amount. Analysis of DNA fragmentsreveals that L1210 produced a ladder of nucleosome sized fragmentsindicative of apoptosis, whereas L1210/DDP cells did not. This latterphenotype—loss in forward scatter and membrane permeability with no “DNAladdering”—is the hall mark of oncosis.

8. Fas Deficient Lymphocytes are also drug resistant to methotrexate: Weisolated splenic lymphocytes from aged-matched wild type C57BL/6 miceand C57BL6.lpr and C57BL.gld. Splenocytes from C57BL/6 lpr or gldanimals were isolated, red cells depleted, and single cell suspensionsprepared. Cells were cultured in the absence or presence of 5×10⁻⁸ Mmethotrexate for 18 or 32 hours. Cells were harvested and viability wasdetermined by flow cytometric analysis and confirmed with trypan blueexclusion.

The data demonstrate decreased susceptibility to methotrexate-inducedapoptosis in Fas deficient lymphocytes. These data are consistent withthe notion that Fas is required for drug susceptibility.

9. Drug resistant cells express intracellular fas, UCP and bcl-2: Wedetermined if wild type and/or drug resistant cells expressintracellular and surface fas, UCP and bcl-2 (FIGS. 1A and 4). Westained non-permeabilized L1210 and L1210/DDP cells for cell surface orintracellular Fas. The data show that while there is no cell surfaceexpression of Fas on the drug/apoptotic resistant cells, the drugresistant cells express high levels of intracellular Fas.

These data show that drug resistant cells are cell surface Fas negativeand protected from death resulting from changes in mitochondrialmembrane permeability transitions.

Example 2 Pancreatic B Cells Express UCP and Have No Cell Surface Fas

1. Loss of antigen in β-cell tumors: Proliferation with two responder Tcell clones, BDC-2.5 and BDC-6.9, was tested using NOD peritoneal cellsas APC and as antigen, either freshly prepared NOD islet cells (control)or β tumor cells, or NIT-1, an established beta tumor cell line from theNOD-RIPTag mouse. Upon harvesting the islet tumors, the β-cells obtainedare fully as antigenic as normal NOD islet cells. The NIT-1 line is alsoantigenic for these T cell clones, but only at low passage numbers; withcontinued culture, the line changes its morphology and growth kineticsand undergoes complete loss of antigen.

2. Response of pancreatic β-cells to glucose: The experiments describedbelow were designed to test the hypothesis that β cell metabolism may belinked to immune recognition and destruction. Glucose utilization wasmeasured as [³H]H₂O production from 5-[³H]glucose in normal rat islets.Glucose oxidation was measured as [¹⁴C]CO₂ production fromU-[¹⁴C]glucose.

The data show increasing glucose utilization and oxidation in β-cells asa function of increasing glucose concentration. FIG. 5 depicts themeasurements for glucose oxidation and utilization (representative ofmany experiments). The expected values of β-cells from normal animals isillustrated in FIG. 5.

FIG. 5 shows glucose utilization and oxidation in normal rat islets.Glucose utilization was measured as [³H]H₂O production from5-[³H]glucose. Glucose oxidation was measured as [¹⁴C]CO₂ productionfrom U-[¹⁴C]glucose.

3. Normal β-cells Express Intracellular UCP2 and No Cell Surface Fas:Normal β-cells have a specialized glucose response which is based on thecell being responsive to physiologic glucose concentrations. The processthat mediates the glucose responsiveness is the process involving fluxthrough glycolysis. β-cell glucose usage is mediated through arelatively unique system that entails specialized high K_(m) glucosetransporter (GLUT2) and glucose phosphorylation isoforms (glucokinase).We isolated β-cells from C3H mice, stained the isolated cells withanti-Fas, and electronically gated viable cells. In parallel, cells werepermeabilized and stained with an antibody to UCP2 (kindly provided byDrs. Jean Himms-Hagen and M. E. Harper).

The results show that normal β-cells express intracellular UCP2 and nocell surface Fas.

4. Fas Expression and Mitochondrial Membrane Potential are a Function ofGlucose Concentration in Mouse β Cells.

The central question is whether Fas expression is altered by changes inphysiological glucose concentrations in normal β cells and does themitochondrial membrane potential increase, suggesting that cell has ATPsynthesis resulting from increased rates of electron transport. Ourdata, FIG. 6, suggest that as glucose concentration, the large β cellsubset of gated cells have increased Fas expression and concomitantincreased mitochondrial membrane potential, while the smaller (possiblyalpha, glucagon producing cells) do not.

In FIG. 6 islets were isolated and dispersed with trypsin and a cellstrainer. Debris and dead cells were removed and applying the cells to a1.066 Percoll gradient. Electronic gating of the cells was used tosegregate the populations of islets cells. The region with larger cellswere gated β cells (13, 14) where the region with smaller cells weregated as alpha cells (13, 14). Other larger cells were excluded becausethey contained δ cells. The cells were treated overnight with eitherphysiological 11.1 mM glucose or high glucose 55.5 mM glucose. Fasexpression was determined by staining with a FITC conjugated antibody.Mean fluorescence of staining with isotype control antibody wassubtracted. Measurement of mitochondrial membrane potential was measuredusing JC-1 as a fluorescence probe (15, 16). The relative membranepotential was read by taking the red mean fluorescence (aggregated JC-1labeled) divided by mean green fluorescent (monomeric JC-1) labeledfluorescence (15, 16).

5. Determination of Mitochondrial Membrane Potential in a Cells Isolatedfrom Four Strains of Animals.

As shown in FIG. 7 mitochondrial membrane potential is assessed flowcytometrically using mitotracker red. The amount of membrane potentialwas measured in the four strains of animals AB-, AB-Ea, C57B1/6, BITgEa,described in more detail below.

Example 3 Relationship Between the Metabolic State of a Cell andExpression of MHC Class II Molecules

1. Analysis of the Mechanisms of Signaling Through MHC Class IIMolecules

i. Perturbation of Class II on Resting B Cells Results in the Generationof cAMP.

Early studies demonstrated that ligation of IE molecules on resting Bcells resulted in the rapid generation of intracellular cAMP in thosecells (Cambier, et al., Nature (1987). Based on this observation and onour more recent evidence that elevated levels of cAMP correlate withdeath in resting B cells, we have studied the generation of cAMP in moredetail. These data are compiled in FIG. 8, panels A, B, C, and D. Thedata in this figure allow a comparative analysis of alterations inlevels of cAMP as induced by antibodies to IE, FIG. 8, panels A and B;mAb to IA (From ATCC), FIG. 8, panels C and D. In FIG. 8B, cells werestimulated with antibodies to MHC class I (From ATCC), with anti-Ig(Jackson Immunochemicals) and L-4 (Genzyme), or with isoproteronol(Sigma) as indicated. In panel C, we isolated B cells from C57BL (whichexpress IA, but not IE molecules) wild type, lpr, or gld animals. Thedata in panel C show that the lpr, or gld mutation does not alter thesignal delivered by MHC class II engagement at the dose of the anti-IAmAb that we have used.

FIG. 8 shows comparative analysis of alterations in levels of cAMP asinduced by antibodies to IE, panels A and B; mAb to IA, panels C and D.In panel B, cells were stimulated with antibodies to MHC class I, withanti-Ig and IL-4, or with isoproteronol as indicated. In panel c, weisolated B cells from C57BL (which express IA, but not IE molecules)wild type, lpr, or gld animals. Dose titration of anti-IA stimulation onB cells from C57BL/6 animals, panel D.

These data show that under the conditions we have employed to stimulatethe cells, the anti-IE antibodies are more effective at increasingintracellular cAMP. To investigate the possibility that the differencesin the ability of IA and IE to alter levels of cAMP may be theconsequence of dose dependent differences and to determine if anti-IAantibodies ever induce increase in cAMP, we performed a dose titrationof anti-IA stimulation on B cells from C57BL/6 animals, (panel D). Thesedata demonstrate an oscillation of cAMP levels, and reflect alterationin signaling by differences in aggregation state of MHC class II.

FIG. 9 shows an analysis of DNA fragmentation from resting B cells. ACells were incubated with medium alone, with anti class II (IA),anti-class II (IA beta chain), anti-class II IE, isoproteronol, ordibutyryl cAMP, lanes 1 through 7 respectively, Panel A, and culturedovernight. Cells were harvested, nuclei separated, and fragmented(non-clear) DNA precipitated and the samples were electrophoresed onagarose gels. Bands were visualized using ethidium bromide andultraviolet light detection. Bands were quantitated using scanningdensitometry, panel B. Quantitated area is presented in panel C.

ii. Anti-Class II mAb Induce an Increase in Apoptotic Cell Death inResting B Cells.

Our data demonstrate that treatment of resting B lymphocytes withanti-class II mAb results in B cell apoptosis, as measured by increasesin nucleosome-sized DNA fragments. These are detected by agarose gelelectrophoresis and quantitated densitometrically. Apoptotic indices aregenerated by comparison to maximum apoptotic death as stimulated byisoproterenol (FIG. 10).

FIG. 10 shows ligation of class II molecules on resting, but notactivated B cells, results in apoptotic death. Resting B cells, (whitebar, black dots); in vitro activated B cells [anti-Ig and recombinantIL-4], (parallel lines bar); or freshly ex vivo activated cells, (blackbar, white dots); were treated with 10 μg/ml anti-1-A^(k) mAb (17/227);anti-Ig and 16 units of recombinant IL-4, or 10:M isoproteronol. B cellswere treated with the stimuli indicated for 10 min at 37° C., culturedovernight, harvested and assayed. (The in vitro activated cells were nottreated with additional anti-Ig plus rIL-4.) Fragmented DNA was isolatedby centrifugation and run on agarose gels. The ethidium bromide stainedgel was photographed and scanned. An Apoptotic Index was calculated bytaking: [(experimental area−area with medium alone)/(area withisoproterenol−area with medium alone)]. Treatments that produce a scoreof 0 show background levels of apoptosis, whereas treatments that areprotective produce scores <0. Data from four independent experiments areaveraged for normal animals; means and standard errors are shown. Meansof two independent experiments for the freshly ex vivo activated cellsand a single experiment for the in vitro activated cells are shown.

iii. MHC Class II Induced Death in Resting B-Cells from Normal MouseStrains, But not Mouse Strains Having the lpr and gld Mutations.

To test the hypothesis that the mechanism of IA-mediated death involvesthe receptor ligand pairs CD95/CD95L, we have used mouse strains thathave the lpr mutation, or the gld mutation, which have defects in CD95and CD95L, respectively (Watanabe-Fukunaga, R, et al., Nature (London),1992, 356, 314-317; Suda, T et al., Cell, 1993, 75:1169-1178). Totalsplenic B-cells were isolated from C3H, AKR, C3H.lpr, and MRL.gld mice.All of these strains are H-2^(k). The cells were cultured overnight,harvested, permeabilized in saponin, stained with propidium iodide (PI)which intercalates into DNA, and analyzed by flow cytometry. After a 15hour culture, a significant percentage of cultured B-cells fragmenttheir DNA, with no stimulation (Newell, M K, et al., Proc Nat Acad SciUSA, 1993, 90:10459-10463). Crosslinking MHC class II IA (HLA-DP/DQ inhumans) on B-cells from the wild type animal cause an increase inapoptosis. Unlike the normal B-cells, there is no increase in less than2×DNA after crosslinking MHC class II on B-cells from lpr or gld mice.

2. Interaction of B-cells with T cells: The results of mAb binding toMHC class II does not, a priori, reflect the result of an interactionwith a physiologically relevant ligand. To address the possibility thatthe physiological ligand for MHC class II is expressed on a CD4+ T cell,we examined the effect of class II signaling resulting from T cell:Bcell interactions, Table 4. Resting splenic B-cells were isolated by Tdepletion and density gradient centrifugation (Percoll). The B-cellswere then combined with either an autoreactive I-Ak-specific T cellhybridoma (Kal-68.4) or with a hen egg lysozyme (HEL) peptide-specific,I-Ak-restricted T cell hybridoma (A6.A₂) either with or without atryptic digest of lysozyme as the source of the required peptide. Cellswere cultured overnight at 37° C. and then examined under a fluorescencemicroscope. Apoptotic cells were scored based on their morphology and ontheir uptake of Hoechst Dye 33342 at 5 μg/ml final concentration (Cohen,J. J, et al., Ann Rev Immun, 1992, 10, 267-293). B and T cells weredistinguished by morphology.

TABLE 4 Induction of apoptosis in resting, but not activated, AKR Bcells by interaction with T cells Resting B cells Activated B cells %Apoptotic IL-2 Titer, % Apoptotic IL-2 liter, Culture Additions ^(a) Bcells U/ml B cells U/ml Medium Alone 14 <20 25 <20 A6.A2 ^(b) 13 <20 18<20 A6.A2 + tryp-HEL ^(c) 54 1280 25 1280 Ka1-68.4 ^(d) 30 160 22 320^(a) Equal numbers (5 × 10⁵) of B-cells and T cells were incubated for16 hr at 37° C. in a 24 well microtiter plate. ^(b) A6.A2 isI-Ak-restricted T cell hybridoma specific for the hen egg lysozymepeptide HEL(aa34-45). IL-2 titers were determined using HT-2 cells aspreviously described (36). ^(c) Tryptic digest of HEL, containingHEL(aa34-45), was used at 1 mg/ml. ^(d) Ka1-68.4 is an autoreactiveI-Ak-specific T cell hybridoma.

3. Phenotypic characterization of apoptotic B-cells: We adapted thetechnique of using terminal deoxynucleotidyl-transferase (TdT) to addfluorochrome-conjugated deoxyribonucleotides to the free ends of DNA toflow cytometric analysis of apoptosis (Gold, R, et al., J HistochemCytochem, 1993, 41:1023-1030). Because the fragmented DNA of apoptoticcells has significantly more free ends that DNA of non-apoptotic cells,the apoptotic cells stain bright green with dUTP-FITC (deoxyuridinetriphosphate) whereas viable cells remain dull. After a 16 hr incubationwith the Kal-68.4 autoreactive T cell hybridoma, resting B-cells fromAKR mice showed 46-47% apoptotic cells, confirming the results using thetwo other methods. This method allows a counter-stain of cells withantibodies directed against various surface receptors expressed by theB-cells.

Relative to resting B-cells, B-cells cultured overnight with Kal-68.4upregulated B7-1, B7-2 and Fas, with the upregulation of B7-1 being themost striking and giving rise to a bimodal distribution (left handpanels, FIG. 11). Two-color analysis reveals that the B-cells from thesecultures may be divided into viable (deoxyuridine-FITC low) andapoptotic (deoxyuridine-FITC high) populations with apparentdifferential expression of the counter-stained receptors on the twopopulations. Histograms of fluorescence intensity of the stainedreceptors (right panels, FIG. 11) show that Fas (FIG. 11 c) andespecially B7-1 (FIG. 11 a) are upregulated on the apoptotic populationwhereas B7-2 (FIG. 11 b) is expressed at higher levels on the viablepopulation.

FIG. 11 shows a two-color flow cytometric analysis of apoptotic restingB-cells. AKR resting B-cells and Kal-68.4 T hybridomas cells wereincubated overnight. Cells were stained with biotin-conjugated mAbdirected against B7-1 (a), B7-2 (b), or Fas (c) followed byPE-streptavidin, and apoptotic cells were detected using TdT/dUTP-FITC.The contour plots on the left of each section show labeling withdUTP-FITC (as a measure of apoptotic death) versus counter-staining withthe indicated mAb for B-cells harvested from culture with T cells. Thepercentages indicate the relative number of cells in the viable(dUTP-FITC dull) and apoptotic (dUTP-FITC bright) populations. Thehistogram on the right of each section shows the relative staininglevels with the indicated mAb of the viable (solid line) and theapoptotic (dashed line) populations as gated by dUTP-FITC fluorescencein the contour plots.

4. Class II-Mediated Signaling in (NZB×NZW)F1 and (NZB×SWR)F1 Mice

i. Engagement of Class II on Resting B-Cells from Autoimmune Strains ofMice does not Result in Increases in cAMP Over Background Levels.

Experiments with B-cells from (NZB×NZW)F1 and (NZB×SWR)F1 mice suggesteda potential link between class II-mediated B cell signaling andautoimmunity in these mice. Following the protocols as described inNewell, M K, et al. (Proc Natl Acad Sci, USA, 1993, 90:10459-10463) forresting B lymphocytes from normal mice, we isolated resting B-cells fromspleens of (NZB×NZW)F1 and (NZB×SWR)F1 mice by Percoll gradientseparation and treated the cells with antibodies to class II molecules,FIG. 12. Experiments utilized mice younger than 3 months of age. Thesemice do not have elevated serum levels of IgG autoantibodies to histoneand DNA and do not demonstrate evidence of an immune-complexglomerulonephritis.

FIG. 12A demonstrates that in contrast to B-cells from normal mice,resting B-cells from (NZB×SWR)F1 and (NZB×NZW)F1 mice do not elevateintracellular cAMP in response to ligation of their class II molecules.In contrast, the response to isoproterenol is normal. It should be notedthat there are no significant differences between resting cells innormal versus (NZB×SWR)F1 mice, as defined by surface MHC class IIexpression and by density (Julius, M and Haughn, L, Eur J Immun, 1992,22, 2323-2329).

FIG. 12A shows class II ligation of resting B-cells from (NZB×NZW)F1 and(NZB×SWR)F1 animals does not result in increases in cAMP. Resting(1.079<r<1.085) B-cells from normal, (NZB×SWR)F1, or (NZB×SWR)F1,animals were treated for 10 min at 37° C. with stimuli indicated. Levelsof cAMP were determined as described in FIG. 1. Means (and standarderrors) from three experiments are shown for normal animals. The meansfrom two experiments are shown for each of the F1 hybrid animals.

ii. Class II-Mediated Apoptosis does not Occur in Resting B Lymphocytesfrom Autoimmune Mice.

We stimulated both resting and activated B-cells from (NZB×NZW)F1 and(NZB×SWR)F1 mice with antibodies to class II molecules. Young animals,prior to the development of lupus-like autoantibody and renal disease,were used in these studies.

Data demonstrates that in contrast to normal B-cells, resting B-cellsfrom (NZB×NZW) F1 and (NZB×SWR) F1 mice are refractory to MHC classII-mediated apoptosis. FIG. 12B also shows that despite normal cAMPgeneration after isoproterenol, this treatment induced minimal evidenceof apoptosis in (NZB×NZW)F1 B-cells. An intermediate level ofisoproterenol-induced apoptosis was apparent in B-cells from (NZB×SWR)F1mice. Thus, the collective results demonstrate that, while restingB-cells from both autoimmune strains are defective in coupling theligation of class II molecules to the generation of cAMP, cells from(NZB×NZW) F1 animals also appear to have a second lesion in theapoptotic pathway that is downstream from the generation of cAMP.

FIG. 12B shows a ligation of class II molecules on resting B-cells from(NZB×NZW)F1 and (NZB×SWR)F1 animals does not result in apoptotic death.Agarose gel electrophoresis of DNA fragments from high density restingB-cells (1.079<r<1.085) was carried out as described in FIG. 2.Normalized areas produced by scanning densitometry on the gels shownabove for normal, (NZB×SWR)F1, or (NZB×NZW)F1, animals. The data werenormalized to background levels rather than calculating an ApoptoticIndex since (NZB×NZW)F1 cells did not show a significant increase inapoptosis when treated with isoproterenol. For normal animals, the meanand standard error of four experiments are shown. For the (NZB×NZW)F1animals, representative data from one of two experiments is shown andfor (NZB×SWR)F1 animals, a single experiment is shown. Becauseexperiments were performed on different days it is impossible to comparebackground levels of apoptosis between strains; however, it should benoted that there is significant variability in background levels ofapoptosis, with those for the (NZB×NZW)F1 animals apparently tending tobe higher than for the other strains.

iii. Phenotypic Characterization of Apoptotic B-Cells from AutoimmuneProne Mice

Our results show that when using B-cells from (NZB×NZW)F1 animalsresting B-cells from these animals are refractory to class II-inducedapoptosis. This indicates that a failure of class II mediated apoptosisprovides a mechanism for polyclonal hyper-gammaglobulinemia,characteristic of autoimmune disease. Resting B-cells are able topresent sufficient self peptides to allow their interaction with the“autoreactive” T cells and that the failure to obtain class II-mediatedapoptosis in resting B-cells may drive polyclonalhyper-gammaglobulinemia.

5. Structural Features and Cell Surface Expression of MHC IA and EMolecules Associated with Cell Death

FIG. 13 shows UCP expression in L1210 and L1210/DDP cells in response tostaurosporin and PMA.

Sequence of MHC IA and MHC IE Molecules

Comparison of the transmembrane (TM) and cytoplasmic (Cy) domainsequences of the beta and alpha chains of IA^(k) and IE^(k) reveals bothconserved and unique sequences. The differences between IA and IE andthe human equivalents are generally shared. The beta chains of IA^(k)and IE^(k) have 18 of 22 amino acids that are conserved in the TMdomain. These changes are basically conserved, whereas the Cy domainsdiffer in length and composition. The Cy domains of IA^(k) has more twomore prolines and an extra two positive charges (R, H) at the proximalend of the Cy domain next to the inner leaflet. The area (RHRSQKGP) (SEQID NO. 13) of the Cy domain of IA^(k) that has been mapped by Wade et al(Int Immunol, 1994, 6:1457-1465) as being required for PKC translocationand cAMP, respectively, is different from the sequence seen in E, butthe residues QKG are the same. This sequence similarities may explainthe observation that ligation of IA or IE can signal increases inintracellular cAMP. The lack of the RH or PP residues in IE couldexplain the lack of Fas induction, due to either the loss of a bindingsite defined by the positive charges of the kinks introduced in the Cydomain by the multiple P residues.

6. Correlation between surface expression and type of cell death. CellSurface Phenotype of MHC Class II⁺ Cells. Much of our work is based uponthe use of model cell lines which have been transfected with wild typeor mutated MHC class II molecules and which exhibit each of theprototypic signaling phenotypes, M12.C3 and K46J, representing cAMP andCa++ generating responses, respectively (Wade, W F et al., Int Immunol,1994 6:1457-1465). The results of flow cytometric analysis to phenotypethe cell surface marker expression on each of these lines is combinedwith a summary of what we know about the way cells die in Table 5. Thesedata were generated either flow cytometrically by assessing changes inforward and side scatter with uptake of ethidium bromide, or bymorphological assessment and trypan blue exclusion.

TABLE 5 Summary of Cell Surface Expression and Cell Death Phenotype Typeof class II induced death (if any) Cell Surface Phenotype Osmotic Fas IEB72 B7-1 Apoptotic Rupture M12.C3 wt/wt++ −− −− + −− ++++ M12.C3 411−12/−18 −− −− −− + −− −− M12.C3 7D3 −12/wt + −− −− + −− + K46J wt/wt +++++ ++ + ++ ++ K46J −12/−18 + + −− + ?? ??

Example 4 Involvement of IA Versus IE in Resistance and Susceptibilityto Immune-Mediated Cardiovascular Disease Coxsackievirus-MediatedMyocarditis

To evaluate the role of MHC class II antigens in immune-mediatedmyocarditis susceptibility, transgenic mice were graciously provided byDr. Chella David of Mayo Clinic. Dr. David supplied the followingstrains: 1) AB^(o) mice lack MHC class II IA and E molecules (class IIknockout (KO) mice); 2). AB^(o) Ea^(b) are MHC class II KO mice whichhave a functional transgenic IE chain, so that the animal express IE butnot IA; 3) Bl.Tg.Ea^(b) mice express the wild type IA molecules as wellas the IE molecules; and 4) wild type C57B16 express IA only.

Male mice, 4-5 weeks of age were injected ip with 100 μg GL3-3A(anti-γδ) monoclonal antibody in 0.5 ml PBS, or PBS alone on days −2 and+2 relative to virus. Animals received 10⁴ PFU CVB3 on day 0 andsurviving animals were euthanized on day 7. Hearts were removed fromanimals between days 5 and 7 for analysis. Hearts were divided and theapex was formalin fixed, sectioned and evaluated by image analysis forpercent of the myocardium affected. The remaining tissue was titered byplaque forming assay for virus. Groups consisted of 4 mice each. Theresults are summarized in Table 6 below.

TABLE 6 Effect of Depleting γδ⁺ T Cells on CVB3-Induced Myocarditis MOR-VIRUS MYO- STRAIN ANTIBODY TALITY TITER CARDITIS C57BL/6 0 5.1 ± 0.7 0.5± 0.3 Anti - γδ TcR 0 5.5 ± 0.9 0 ± 0 AB⁰ 0 6.5 ± 1.4 0 ± 0 Anti - γδTcR 0 7.1 ± 0.8 1.3 ± 0.8 AB⁰ Eαk 100 6.2 ± 0.9 5.1 ± 2.0 Anti - γδ TcR25 6.5 ± 0.7  1.8 ± 1.1* B1 Tg Eαk 50 4.3 ± 0.5 8.3 ± 1.6 Anti - γδ TcR0  5.3 ± 0.4*  1.7 ± 0.5* *Significantly different thannon-antibody-treated mice at P ≦ 0.05.

Mice expressing either no class II MHC antigen or IA only weremyocarditis resistant having little or no cardiac inflammation and noanimal mortality. In contrast, IE-bearing mice showed increasedmortality accompanied by substantial myocardial necrosis. AB^(o) Eαmice, expressing IE only, began dying earlier (day 3 post-infection) andhad more extensive coaggulative myocardial necrosis with limited cardiacinflammation compared to Bl.Tg.Eα mice (both IA+ and IE+). Cardiaclesions in Bl.Tg.Eα mice were confined to regions of mononuclear cellinfiltration and were characterized by extensive myocyte dropout. Viraltiters also differed between mouse strains with the highest titersoccurring in AB^(o) and AB^(o) Eα mice. This suggests that IA expressionis important in virus clearance. Also, the elevated viral titers inAB^(o) Eα mice must not be directly responsible for the necrotic heartlesions in this strain since AB^(o) mice also have elevated virusconcentrations but no histological evidence of cardiac injury. Thus, byeither animal mortality or histology, the presence of E in C57BL/6 miceaggravated CVB3-induced disease. Treating the BL tg Eα^(k) strain withantibodies to deplete γδ T cells conferred resistance to myocarditis. Wemeasured cytokine profiles from total splenocytes of the animals beforeand after infection. We observed increased γ-interferon in all strainsand higher levels of L4 in the Bl.Tg.Eα^(k) animals. Depletion of γδ Tcells resulted in an increased percentage of cells producing IL4 postinfection (not shown). This result demonstrates that the cytokine biasis important in the development of myocarditic lesions.

Example 5 MHC Class II IE Molecules Confer Protection from EarlyAtherosclerotic Fatty Lesions

Several studies suggest that CD4⁺T lymphocytes contribute to thepathogenesis of fat-induced atherosclerotic lesions (Emeson, E E, etal., Am J Path, 1996, 149:675-685). We addressed the possibility thatexpression of IA and/or IE impacted the development of lesions whichresult from a high fat diet.

C57BL/6 transgenic mice differing in MHC class II antigen expressionwere kindly supplied by Dr. Chella David (Mayo Clinic). Between 4 and 10mice of each strain were placed on high-fat, high-cholesterol diet(Teklad #96354; 20% total fat, 1.5% cholesterol, 0.5% sodium cholate) atthree weeks of age and were killed 15 weeks later for evaluation of theaorta and splenocytes. An additional group of 7 C57BL/6 mice were placedon high-fat diet as above, but were injected ip every two weeks with 100μg monoclonal rat anti-CD4 antibody (clone GK1.5; American Type TissueCollection, Bethesda, Md.). This protocol has been used previously tomaintain CD4+ T cell-deficient mice for extended periods in theexperimental allergic encephalomyelitis (EAE) model. The heart andascending aorta including the aortic arch were removed and evaluated foratherosclerotic lesions according to the method of Plump et al. usingoil red-0 stained serial sections. Briefly, hearts were fixed in 10%buffered formalin, embedded in 25% gelatin, grossly cut through theventricles parallel to the atria, frozen in OCT and sectioned bycryostat. Ten micron thick sections were placed on 5% gelatin coatedslides, stained with 0.24% oil Red-0 (neutral lipids) and counterstainedwith 2.4% hematoxylin (nuclei and basophilic tissue) and 0.25% lightgreen (remaining tissue). Lesions were quantified by area morphometryusing a compound light microscope.

TABLE 7 MHC class II IE molecules confer protection from earlyatherosclerotic fatty lesions Fatty Lesion Strain IA IE Treatment HeartLiver Cholesterol C57BL/6 + − No ++ − Not Treatment (++ Lymph)Significant C57BL/6 + − Anti-CD4 + − Not Significant AB⁰ − − No +/± +++Not Treatment Significant AB⁰Eαk − + No −/± +++ Not TreatmentSignificant B1TgEαk + + No −/± − Not Treatment (++ Lymph) Significant

Our data demonstrate that the presence of MHC class II IA correlatedwith susceptibility to fatty lesions in the hearts of the C57BL/6animals and that the presence of IE molecules conferred protection fromthe fatty lesions. The role of CD4 cells in this process was confirmedby the finding that removal of CD4 cells from the susceptible C57BL/6abrogated the pathology in the heart. Note the correlation betweenexpression of IE, increased production of L-4, and protection from fattylesions resulting from a high fat diet. The results in Table 7demonstrate that CD4+T cells contribute to early fat deposition in theaortic sinus and that IE molecules suppress lesions. MHC class IImolecules, IA or IE, regulate susceptibility to development of earlyatherosclerotic plaques, and that cytokine profiles are altered (notshown).

Example 6 NGF and EGF-Dependent Changes in Fas (CD95), B7.1 (CD80) andB7.2 (CD86) Expression on PC12, TrkA, and v-Crk Neuronal Cell SurfaceMaterials and Methods

Cell Culture: Rat pheochromocytoma cell lines, including PC12, Trk andv-Crk cells were a kind gift from Dr. Raymond Birge. They weremaintained in complete RPMI 1640 (GIBCO) supplemented with 7% heatinactivated fetal calf serum and 3% heat inactivated horse serum at 37°C. in a humidified incubator with 5% CO2. The PC12 transfectants weregenerated as described previously (Glassman et al., 1997, Hempstead etal., 1994). Cells were plated in 6 well culture plates at aconcentration ranging from 2-8×106 cells/well, according to the celltype's growth kinetics, and 5 ml complete medium. NGF-7S from mousesubmaxillary glands (Sigma Chemical Co.) or EGF, kindly provided by Dr.Raymond Birge, was added at a concentration of 50 ng/ml culture mediumand the cells were incubated for 24 or 48 hours.

Cytofluorometric Analysis. Cells were harvested after a 10 minuteincubation period on ice in order to diminish their adherence to theplastic culture flask. They were spun at 1210 rpm, for 7 minutes,resuspended in medium and counted after trypan blue staining. Equalnumbers of cells were placed in 12×75 mm flow tubes (range: 0.1-1×106cells/tube), washed in PBS and 5% FBS and stained at 4° C. The followingstains were used: a) fluorescein isothiocyanate (FITC) hamster IgGisotype standard, b) FITC anti-mouse Fas, c) FITC anti-mouse CD80(B7.1), d) phycoerythrin (PE) mouse IgG2b K isotype standard and e) PEanti-mouse CD86 (B7.2). All antibodies were obtained from Pharmingen.

After incubation on ice for 20 minutes, cells were washed, resuspendedin PBS and 5% FCS and analyzed by flow cytometry (Becton Dickinson).Histogram plots were derived from dot plots gated on the live cellpopulation according to the forward versus side scatter ratio (CellQuest Program). The absolute Fas, B7.1 and B7.2 values presented on thegraphs were obtained by subtracting the geometric mean fluorescence ofthe specific antibody from the mean geometric fluorescence of thecorresponding isotype. Values were considered subjectively positive (+)if the difference was statistically significant (p<0.001) according tothe applied Kilmogorov-Smirnov statistical analysis.

Results

Fas cell surface expression after NGF stimulation: As shown in FIG. 25(panels A, D, G) Fas is constitutively expressed on the surface of PC12and TrkA cells. NGF stimulation abrogates Fas expression on PC12 cellsand paradoxically increases its expression on Trk cells at 24 hours. Faslevels tend to return to basal values on PC12 cells and are maintainedconstant on TrkA cells after 48 hours of NGF stimulation. V-Crkconstitutive cell surface Fas expression is minimal but statisticallysignificant, and it totally abrogated after NGF stimulation at 48 hours.

Fas cell surface expression after EGF stimulation: EGF stimulation at 24and 48 hours also downregulates Fas expression on PC12 and Trk cells;EGF stimulation at 48 hours totally abrogates Fas expression on bothcell types. On the other hand, EGF stimulation at 24 hours significantlyup-regulates Fas expression on v-Crk cells, but it is againdown-regulated and abrogated by EGF stimulation at 48 hours.

B7.1 cell surface expression after NGF stimulation. B7.1 isconstitutively highly expressed on the surface of unstimulated PC12 andTrk cells (FIG. 25, panels B and E). Its expression is minimal onunstimulated v-Crk cells (FIG. 25, panel H). NGF stimulation initiallydownregulates B7.1 expression on PC12 cells at 24 hours, but it tends toreturn to basal values at 48 hours. NGF stimulation has no effect onB7.1 expression on the surface of Trk cells and v-crk cells.

B7.1 cell surface expression after EGF stimulation: EGF stimulation at24 hours significantly lowers the detection of high B7.1 levels on thesurface of PC12 cells; EGF stimulation at 48 hours reestablishes thebasal values. As in PC12 cells, B7.1 expression is lowered after EGFstimulation of Trk cells at 24 hours and reestablished at the 48 hourtime point. EGF stimulation significantly increases B7.1 expression onv-Crk cells after 24 and 48 hours of culture.

B7.2 cell surface expression after NGF stimulation: B7.2 cell surfaceexpression is minimal on unstimulated PC12 cells (FIG. 25, panel C,F,I).NGF stimulation at 24 hours has no effect on its expression butstimulation at 48 hours completely abrogates its expression. Trk B7.2cell surface expression is also minimal on unstimulated cells, it isslightly down-regulated by NGF stimulation at 24 hours and abrogated byNGF withdrawal. There is no B7.2 cell surface expression on v-Crk cellsnor is it induced by NGF stimulation.

B7.2 cell surface expression after EGF stimulation: EGF stimulation at48 hours up-regulates B7.2 expression on PC12 but this is mostsignificant on the surface of Trk and v-crk cells. EGF stimulating at 48hours is the only instance whereby there is significant induction of theconstitutively absent B7.2 molecule on v-Crk cells. EGF withdrawaltotally rescinds this effect and B7.2 levels are again undetectable.

In PC12 cells and its mutant variants, NGF induces proteins required forthe acquisition of a sympathetic neuronal phenotype, potentiatingcellular differentiation as reflected by an increase in the size andflattening of the neuronal soma and particularly by inducing neuriteoutgrowth (Ray Paper, J. Bio Chem 1995). By contrast, EGF stimulation ofthese cells induces their entry into the cell cycle and thus, cellularproliferation, by binding to another receptor also belonging to thetyrosine kinase receptor family (Hempstead et al., 1994; Siegel et al.,1994). The NGF and EGF receptor pathways appear to be very similar sincethey both activate the receptor-type tyrosine kinases, the Erk2/MAPKpathway and involve the Ras proteins (Chao, 1992; Ray, Id MenendezIglesias et al., 1997) It has been found, according to the invention,however, that the effects of these molecules are quite different on theinduction and abrogation of Fas, B7.1 and B7.2 expression on theneuronal cell surface suggesting that the induction of these moleculesby growth factors NGF and EGF is mediated by a different intra-cellularsignaling pathways albeit dependent on tyrosine kinase activation.

As described above Fas B7.1 and B7.2 are constitutively expressed onPC12 and TrkA cells maintained in culture. Fas expression on PC12 cellsis significantly decreased by NGF stimulation (NGFS). Early NGFS of TrkAcells induces an increase in Fas expression over basal levels. Microgliaconstitutively express Fas ligand (Bonetti and Raine, 1997; MenendezIglesias et al., 1997) and perhaps direct cell-cell contact between theneurons and microglia is required for the interaction of Fas and Fasligand and the development of apoptosis or a co-stimulatory mitoticsignal. In TrkA cells that overexpress the Trk receptor, NGF-induced Fasexpression can promote cell division as NGF stimulates mitosis at thattime period and synchronously play a role in the differentiation processand the development of filopodia. On TrkA cells, the higher induction ofFas expression correlates with the increased numbers of tyrosine kinaseA surface receptors and thus, the development of a sustained increasedstimulus for the mRNA translation of the Fas protein and its secondaryexpression on the cell surface.

EGF stimulation (EGFS) significantly diminishes and even abrogates Fasexpression on PC12 and TrkA cells. However, its expression increasesthree-fold in v-Crk cells transiently after EGFS at 24 hours anddisappears after EGFS at 48 hours. It has been shown that EGFS inducesthe development of neurite processes on the PC12 v-crk mutant not onnative PC12 cells (Hempstead et al., 1994) and this clearly correlateswith the induction of Fas on the v-Crk membrane cell surface, but doesnot explain the down-regulation of Fas observed in Trk and PC12 cells.EGF receptors are also expressed on cortical neurons, the cerebellum andhippocampus, and appear to act on mitotic cells and postmitotic neurons(Yamada et al., 1997).

NGFS and NGFW condition a minor upregulation of B7.1 on PC12, TrkA andv-Crk cells; however, NGFW at 48 hours does diminish B7.1 expression by60% on v-Crk cells. In contrast, we consider that the effect of NGF onB7.2 on all three cell types is negligible. However, EGFS at 48 hours,significantly increases B7.1 expression on all cell variants but itsexpression clearly decreases after EGFW. EGFS at 48 hours alsosignificantly increase B7.2 expression on PC12 and Trk cells and inducesits expression on v-Crk cells. EGFW at 24 and 48 hours correlates withB7.2 down-regulation in PC12 and TrkA cells and paradoxically increasesB7.2 expression no v-Crk cells at 24 hours; it is immediatelydown-regulated after EGFW at 48 hours. Therefore, Fas expression onv-Crk cells and B7.1-B7.2 expression on all-cell types, but particularlyon v-Crk cells, appear to be EGF-dependent. V-Crk cells arecharacterized by the presence of a fusion protein with viral gagsequences fused to the cellular sequences of the Src homology regions 2and 3 (SH2 and SH3) (Hempstead et al., 1994). C-src also possessestyrosine kinase activity (Vaingankar and Martins-Green, 1998) andperhaps these modifications in its sequence allow it to act similarly tothe EGF receptor per se and increase the signal intensity for theexpression of these cell surface molecules. Withdrawal of the stimulus(EGFW) reverts the expression of these molecules towards basal levels.

Identification of Immune Recognition Molecules on Treated VersusNon-Treated Ganglia.

Ganglia, are removed from Po (one-day old mice) brain and plated intocultures. The sensory neurons do not have to be separated away fromSchwann cells. Isolated ganglia are cultured for at least 72 hours underthe following conditions:

1) No Treatment

2) In the presence of nerve growth factor (NGF) for 56 hours

3) followed by harvest, wash, and replating in the presence ofantibodies to NGF. Cells are harvested by cell scraping and dispersedinto single cell suspensions. Cells are stained for cell surface B7-1,B7-2, CDR8, and Trk A (NGF receptor) using monoclonal antibodies tothese molecules.

The cells are cultured as described above but in the presence ofCTLA-4-HuIg to inhibit cell interactions (synapses) which will protectGroup I from death. This shows that B7-bearing cells cause the CD28+ orCTLA4+ cell to release NGF and promote innervation. Additionallyhistological sections are stained by immunofluorescence (using the antiB7 and TrkA antibodies) immediately ex vivo intact mouse brain.

Example 7 UCP is Present in a Panel of Tumor Cells

We extended our analysis of intracellular expression of UCP to othertumor cells (FIG. 14). All of the tumor cells lines examined express UCPintracellularly. These data are consistent with the possibility thatexpression of UCP in tumor cells is generalizable to all tumor cells,and likely results from the well documented shift in subcellularproduction of ATP from mitochondria to cytosol as cells divide.Importantly, these data also demonstrate that expression of UCP2 is notspecific to lymphoid tumors. The L929 cells are fibroblasts and the PC12Trk cells which are derived from pheochromocytoma cell lines,respectively. The EL4 cells are a mouse thymoma cell line and Jurkat arehuman T cell tumor cells. Flow cytometric analysis of intracellular UCP.Isotype control (thin lines) versus anti-UCP (thick lines), on cellswhich had been permeabilized and stained as indicated. The histogramsrepresent FITC isotype control (thin) versus stained with Rabbitanti-UCP (a kind gift of Mary Ellen Harper) FITC-anti-Rabbit outerstep(thick lines). A Coulter Epics Elite flow cytometer with a singleexcitation wavelength (488 nm) and band filters for PE (575 nm), FITC(525 nm) and Red613 (613 nm) was used to analyze the stained cells. Eachsample population was classified for cell size (forward scatter) andcomplexity (side scatter), gated on a population of interest andevaluated using 40,000 cells. Criteria for positive staining wereestablished by comparison with isotype controls, thin lines to specificstain, thick lines.

To confirm that flow cytometrically detected UCP expression wasmitochondrial, we isolated mitochondria from L1210 and L1210 DDP, andperformed Western Blot analysis blotting with rabbit anti-UCPantibodies, FIG. 15. This representative blot shows greater levels ofmitochondrial UCP in the drug resistant L1210/DDP than in L1210/0. Thedetected mitochondria protein has an approximate molecular weight of 30kDa, close to the predicted molecular weight of UCP2 (33 kDa).

Representative Western blot of protein isolated from purifiedmitochondrial fractions of L1210/0 and L1210/DDP cells. Mitochondriawere isolated using differential centrifugation as adapted from REF, REF(Reinhart, P H, Taylor, W M and Bygrave F L (1982) A procedure for therapid preparation of mitochondria from rat liver. Biochem. J. 204:731-735, and Sims N R (1990) Rapid isolation of metabolically activemitochondria from rat brain and subregions using Percoll densitygradient centrifugation. J. Neurochem. 55:698-707.) Lane 1: Molecularweight markers (BIORAD Biotinylated SDS-PAGE standards. After transferof proteins to nitrocellulose, this lane is cut off and detection ofstandards is performed using Avidin-HRP). Lanes 2 and 3: L1210/0mitochondrial protein (40:g) from two distinct mitochondrialpreparations. Lanes 4 and 5: L1210/DDP mitochondrial protein (40:g) fromtwo distinct mitochondrial preparations. Lane 6: uncoupling proteinstandard (0.75:g) from rat brown adipose tissue (which expresses UCPs1-3). Rabbit anti-hamster UCP was used at a dilution of 16,000. Thesecondary antibody: goat anti-rabbit IgG conjugated to HRP at 1:10,000.Chemiluminescent detection: Amersham ECL kit.

To determine whether increased UCP corresponded to increasedmitochondrial proton leak and a lower mitochondrial membrane potential(ΔQm) we assessed characteristics of non-phosphorylating respiration inintact L1210 wild type and L1210 DDP cells (FIG. 16). State 4 ΔQm in DDPcells, x mV, was significantly lower than in wild type cells, y mV(p<0.001), and state 4 oxygen consumption in DDP cells is significantlyhigher than in wild type cells, indicating increased mitochondrialproton leak.

Example 8 Rates of Glucose Utilization, Oxidation and Cell Surface andIntracellular Fas Levels in Melanoma Cells

FIG. 17 depicts the level of cell surface Fas expression onnon-permeabilized (panel A) and intracellular Fas expression inpermeabilized (Panel B) B16 melanoma cells. B16 cells were cultured inthe in the presence of different concentrations of sodium acetate andFas expression was measured. With increasing concentrations of sodiumacetate, the levels of intracellular Fas declined and the levels of cellsurface Fas increased, demonstrating a translocation of Fas fromintracellular stores to the surface.

FIG. 18 depicts the rates of glucose utilization and oxidation in B16melanoma cells. Again cells were cultured in the presence of varyingconcentration of sodium acetate. Both glucose utilization and glucoseoxidation (measured in nmoles) decreased with increasing concentrationsof sodium acetate, demonstrating a correlation with expression of cellsurface Fas in the same cells.

Example 9 Normal Mouse T Cells Express IE

Previous work with human T cells indicates that activation of the Tcells by antigens or engagement of CD4 results in expression of HLA DRon the T cell surface. Expression of MHC class II on mouse T cells iscontroversial. Reports indicate both positive and negative results. Thestudies to date did not distinguish between failure to express IA versusIE. To address the possibility that normal mouse T cells express E weisolated lymph nodes or spleens as indicated from strains of animalswhich express IE, Balb/c, CBA, and AKR mice, spleens or nodes were takenfrom 4 week old mice, minced to single cell suspension, and red bloodcells were removed via Gey's treatment. Splenocytes were then passedover Cellect Columns (Cytovax, Edmonton, Canada) to purify CD4⁺ T cells.CD4⁺ T cells were collected, found to be 98.5% pure, and contaminantswere identified as NK and γδ T cells flow cytometrically. The CD4 Tcells were treated with antibody to CD4 (GK1.5) at 10 μg/ml/10⁷ cellswashed and treated with rabbit anti-rat antibody for 45 minutes at 37°C., followed by washing. The cells were cultured overnight and stainedwith FITC conjugated anti-IE antibody (14-4.4 S), FIG. 19, or 14-4.4Sand counterstained with anti-TCR as indicated in FIG. 19 b.

For the PCR experiment below, FIG. 20 purified CD4⁺ T cells, 5×10⁶/ml,were incubated for 8 hrs. with biotinylated antibodies for CD4 (GK1.5),CD28, CD3 (145.2C11) alone, CD4 and CD28, CD3 and CD28, or no treatment.Experimental setup included wells of purified T cells and percollisolated B cells added to control for potential MHC Class II⁺contaminants. B cells, 5×10⁵ cells, which is 5% of purified T cells (fargreater than the 1.5% contaminants seen following Cellect Columnpurification) were added to T cell wells. Cells were then washed,collected and total RNA was isolated using an RNA isolation kit, RNEasy(Qiagen, Chatsworth Calif.). Single strand DNA was generated from 2*g ofRNA using SuperScript II reverse transcriptase (GIBCo/BRL Gaithersburg,Md.). PCR was done using MHC Class II, (1-E, exon 35′-TAGCTGAGCCCAAGGTGACT SEQ ID NO 14 and 5′ TCACCAGGGTCTGGTAGGTC SEQ IDNO 15) primers. PCR protocol was: 1 min at 94° C., 1 min at 60° C., and2 min. at 72° C. for 35 cycles. Following PCR, samples were loaded onto1% agarose gels, stained with ethidium bromide and visualized with UVlight.

Example 10 Use of Fatty Acids as a Mitochondrial Carbon Source

FIG. 21 shows the results of fatty acid (Oleic Acid) as a mitochondrialcarbon source. Rate of oleate oxidation was measured by incubating cellsfor 90 min at 37° C. in 100 μl of reaction buffer, glucose (2.8, 8.3,27.7 mmol/l), 1.7 mCi (U-14C oleic acid). The reaction was carried outin a 1 ml cup in a 20 ml scintillation vial capped by a rubber stopperwith a center well that contains filter paper. Metabolism was stoppedand CO₂ liberated with 300 μl 1 mol/l HCl injected through the stopperinto the cup containing the cells. CO₂ was trapped in the filter paperby injecting 10 ml 1 mol/l KOH into the center well, followed 2 hourslater by liquid scintillation counting. Tubes containing NaHCO₃ and nocells were used to estimate the recovery of ¹⁴CO₂ in the filter paper,routinely close to 100%. Values indicate the rate of CO2 production byL1210DDP cells (round symbols) or L1210 cells (square symbols). TheL1210 DDP use oleic acid at much higher rates than the L1210 cells.

Example 11 cAMP Levels in L1210 and L1210DDP

FIG. 22 shows levels of cAMP in L1210, left panel versus L1210DDP, rightpanel. Increasing intracellular levels of cAMP are necessary for theactivity of uncoupling proteins. We have shown that class II engagementresults in increased cAMP and we have determined that the mitochondrialmembrane potential of L1210DDP cells is lower than L1210 cells. Thus, weused a radioimmunoassay to determine the levels of cAMP in L1210, leftpanel versus L1210DDP, right panel. Cells were treated for 10 minuteswith nothing, antibodies to IA, IE, or with a beta adrenergic agonist,isoproterenol (10 microMolar). Cells were harvested and cAMP wasextracted from the cells and cAMP levels determined using 125 I labeledcAMP in competitive inhibition in the presence of antibodies to cAMP,radioimmunoassay.

Example 12 Sodium Acetate as a Mitochondrial Modifying Agent

FIG. 23 is a graph depicting Sodium Acetate as a mitochondrial modifyingagent. L1210 or L1210DDP cells were cultured in the presence of gradedconcentrations of sodium acetate in the medium. Cells were stained withJo2.2, a fluorescein conjugated anti-Fas antibody, or an isotypecontrol. Cell surface staining was measured flow cytometrically. ThePercentage of mean fluorescence intensity over the isotype control wasplotted. The data indicate that the presence of acetate increases cellsurface Fas expression in both cell lines.

FIG. 24 is a graph depicting the effects of acetate on susceptibility toFas-dependent cell death. Cells cultured with acetate were loaded with51 Cr and plated onto FasL bearing or mock transfected fibroblast todetermine sensitivity to Fas-induced cell death. Results are reported aspercent chromium release from cells in the presence of FasL bearingcells over mock-transfectants. The data indicate that in a dosedependent manner, culture of both cell types with acetate results insusceptibility to Fas-dependent cell death.

Each of the foregoing patents, patent applications and references ishereby incorporated by reference, including U.S. Provisional ApplicationSer. No. 60/082,250 filed Apr. 17, 1998, U.S. Provisional ApplicationSer. No. 60/101,580 filed Sep. 24, 1998 and U.S. Provisional ApplicationSer. No. 60/094,519 filed Jul. 24, 1998, from which this applicationclaims priority under 35 USC § 119(e). While the invention has beendescribed with respect to certain embodiments, it should be appreciatedthat many modifications and changes may be made by those of ordinaryskill in the art without departing from the spirit of the invention. Itis intended that such modification, changes and equivalents fall withinthe scope of the following claims.

1. A method for inducing apoptosis in a tumor cell, comprising:contacting a tumor cell with an amount of a metabolic modifying agent,which when exposed to a cell causes coupling of electron transport andoxidative phosphorylation, effective to increase the mitochondrialmembrane potential in the tumor cell, wherein the metabolic modifyingagent is selected from the group consisting of glucose, an MHC class IIHLA-DP/DQ ligand, phorbol myristate acetate in combination withionomycin, GDP, sodium acetate, UCP antisense, dominant negative UCP,and staurosporine, and contacting the tumor cell with an amount of anapoptotic chemotherapeutic agent effective for inducing apoptosis in thetumor cell.
 2. The method of claim 1, wherein the apoptoticchemotherapeutic agent is selected from the group consisting ofadriamycin, cytarabine, doxorubicin, and methotrexate.
 3. The method ofclaim 1, wherein the metabolic modifying agent and the apoptoticchemotherapeutic agent are administered simultaneously.
 4. The method ofclaim 1, wherein the metabolic modifying agent and the apoptoticchemotherapeutic agent are administered locally.
 5. The method of claim1, wherein the tumor cell is resistant to the apoptotic chemotherapeuticagent.
 6. The method of claim 1, wherein the tumor cell is sensitive tothe apoptotic chemotherapeutic agent, and wherein the amount ofmetabolic modifying agent is effective to increase mitochondrialmembrane potential and the amount of apoptotic chemotherapeutic agent iseffective to inhibit the proliferation of the tumor cell when themitochondrial membrane potential is increased.
 7. A method for inducingapoptosis in a tumor cell, comprising: contacting a tumor cell with anamount of a metabolic modifying agent, which when exposed to a cellcauses coupling of electron transport and oxidative phosphorylation,effective to increase the mitochondrial membrane potential in the tumorcell, and contacting the tumor cell with an amount of an apoptoticchemotherapeutic agent effective for inducing apoptosis in the tumorcell, wherein the apoptotic chemotherapeutic agent is selected from thegroup consisting of cytarabine, doxorubicin, and methotrexate.
 8. Themethod of claim 7, wherein the metabolic modifying agent is selectedfrom the group consisting of glucose, an MHC class II HLA-DP/DQ ligand,phorbol myristate acetate in combination with ionomycin, GDP, sodiumacetate, UCP antisense, dominant negative UCP, and staurosporine.
 9. Themethod of claim 7, wherein the metabolic modifying agent and theapoptotic chemotherapeutic agent are administered simultaneously. 10.The method of claim 7, wherein the metabolic modifying agent and theapoptotic chemotherapeutic agent are administered locally.
 11. Themethod of claim 7, wherein the tumor cell is resistant to the apoptoticchemotherapeutic agent.